Team:Potsdam Bioware/Labjournal/July

From 2011.igem.org

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Latest revision as of 21:15, 18 September 2011

1 28th Labday 2011-07-01
- 1.1 Agarose gel electrophoresis of PCR (mdnA for phage display, strategy 1)
- 1.2 purification of PCR product mdnA SfiI for phage display
- 1.3 Agarose gel electrophoresis of myc-tagged mdnA
2 29th Labday 2011-07-04
- 2.1 digestion of PCR product mdnA with restriction enzyme SfiI (strategy 1)
- 2.2 ligation of mdnA into pak100(strategy 1)
- 2.3 Amplification of mdnA for phage display (strategy 2)
- 2.4 Gel electrophoresis PCR product mdnA (strategy 2)
3 30th Labday 2011-07-06
- 3.1 digestion of PCR products mdnA, geneIII and vector pARW089 (strategy 2)
- 3.2 Gel extraction from agarose gel of digested fragments mdnA, geneIII and pARW089 (strategy 2)
4 31th Labday 2011-07-07
- 4.1 repeated digestion of pARW089 with NarI and AatII (strategy 2)
- 4.2 Gel electrophoresis of digested pARW089 vector(strategy 2)
- 4.3 ligation of mdnA and geneIII into pARW089(strategy 2)
5 32th Labday 2011-07-12
- 5.1 Transformation of ligation mdnA, geneIII, and pARW089 (2011-07-07) for phage display (strategy 2)
6 33th Labday 2011-07-13
- 6.1 Error prone PCR (varied MnCl2 concentration) of mdnA
7 34th Labday 2011-07-14
- 7.1 Agarose gel electrophoresis of error prone PCR of mdnA from 2011-07-13 and gel extraction
- 7.2 Digestion of error prone PCR of mdnA from 2011-07-13 and pARW089 and agarose gel electrophoresis
- 7.3 Overnight culture of PDV089
- 7.4 Repeated PCR of mdnA for phage display (strategy 1) repeated with new ordered reversed primer (with myc-tag) and geneIII
8 35th Labday 2011-07-15
- 8.1 Ligation of mutated mdnA into pARW089
- 8.2 Transformation of competent E. coli cells with mutated mdnA in pARW089
- 8.3 ligation of mdnA into pak100(strategy 1)
- 8.4 Transformation of ligation mdnA into PAK100 bla KDIR for phage display (strategy 1)
- 8.5 ligation of mdnA and geneIII for strategy 2
- 8.6 ligation of the fusion gene mdnA-geneIII into pARW089(strategy 2)
9 36th Labday 2011-07-16
- 9.1 Transformation of ligated pARW089 with mdnA-geneIII
- 9.2 Overnight culture of XL1 blue with mdnA in PAK100blaKDIR
10 37th Labday 2011-07-17
- 10.1 Miniprep of E. coli overnight culture containing PAK100blaKDIR
- 10.2 E. coli XL1 blue stock culture containing PAK100blaKDIR with mdnA (Phage Display-strategy 1)
- 10.3 overnight culture of E. coli Xl blue m. Ligation089jessi, u.Ligationvector089jessi,u.Ligationu.BandemdnAgeneIIIpARW089, mLigatiojnm.BandemdnAgeneIIIpARW089
11 38th Labday 2011-07-18
- 11.1 overnight culture of E. coli XL-1 blue containing mutated mndA in parW089
- 11.2 Dual-Tranformation of XL1-blu (E. coli) with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla
- 11.3 Test digestion of ligations for strategies 1 and 2
12 39th Labday 2011-07-19
- 12.1 Resistance test of the Dual-Transformation of XL1-blue (E. coli) with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla
- 12.2 Stock cultures of XL1-blue(E. coli)with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla and of XL1-blue(E. coli)with pTEV-SCS1 and of XL1-blue(E. coli)pJC354_ssTorA_NheI_CS-TEV_XhoI_bla
- 12.3 Design of Primers for BioBrick mdnA and mdn-Cluster
- 12.4 Miniprep of E. coli Xl blue containing mutated mndA in parW089
13 40th Labday 2011-07-20
- 13.1 Resistance test of the Dual-Transformation of XL1-blue (E. coli) with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla
- 13.2 Restriction enzyme digestion pARW089 and pARW071
- 13.3 overnight culture of E. coli Xl blue m. ligation mdnA and geneIII into pARW089
- 13.4 Repeated PCR of mdnA and gene III for phage display (strategy 1+2)
- 13.5 digestion of PCR products and vectors (strategy 1+2)
- 13.6 ligation of mdnA and geneIII into digested pARW089(strategy 2)
- 13.7 ligation of mdnA into pak100blaKDIR (stategy 1)
14 41th Labday 2011-07-21
- 14.1 Test digestion of ligation for strategy 2
- 14.2 Transformation of ligated pARW089 with mdnA-geneIII and ligated PAK100 bla KDIR with mdna
15 42th Labday 2011-07-22
- 15.1 Restriction enzyme digestion pARW089 and pARW071 AND trouble-shouting
16 43th Labday 2011-07-23
- 16.1 Restriction enzyme digestion pARW089 (different incubation times), plasmid purification and ligation with EP-PCR mdnA
- 16.2 creation of pPDV089 in Genious
- 16.3 sending pPDV089 from positive clone to GATC for sequencing
17 44th Labday 2011-07-24
- 17.1 Transform ligation from 42th Labday into E. coli XL blue 1 cells
18 45th Labday 2011-07-25
- 18.1 overnight culture of E. coli XL-1 blue containing mutated mndA in parW089
19 46th Labday 2011-07-26
- 19.1 Miniprep of E. coli Xl blue containing mutated mndA in parW089
- 19.2 Alignment of sequenced pPDV089 and model of pPDV089
- 19.3 Repeated PCR of mdnA and gene III for phage display (strategy 1+2)
- 19.4 digestion of PCR products and vectors (strategy 1+2)
- 19.5 gel electrophoresis of digested fragments and vectors
- 19.6 Repeated PCR gene III for phage display (strategy 2)
20 47th Labday 2011-07-27
- 20.1 digestion of PCR product geneIII (strategy 2)
- 20.2 gel electrophoresis of digested geneIII
- 20.3 ligation of mdnA into pak100blaKDIR (stategy 1)
- 20.4 Transformation of generated pPDV100 (strategy 1)
21 48th Labday 2011-07-28
- 21.1 overnight culture of 10 picked clones of XL blue cells transformed with pPDV100
- 21.2 ligation of mdnA and geneIII (strategy 2)
- 21.3 ligation of mdnA/geneIII into digested pARW089(strategy 2)
- 21.4 Transformation of generated pPDV089 (strategy 2)
22 49th Labday 2011-07-29
- 22.1 Miniprep of E. coli overnight culture containing pPDV100
- 22.2 gel electrophoresis of digested pPDV100
- 22.3 creation of pPDV100 in Genious

28th Labday 2011-07-01

Agarose gel electrophoresis of PCR (mdnA for phage display, strategy 1)

Investigators: Sabine, Sandrina

Aim: Verification of mdnA for phage display (strategy 1)

Time: 2011-06-30,14:00-15:30

Materials:

Agarose broad range (Roth)

1x TAE buffer

Gel Red

PCR products: mdnA for phage display (strategy 1) from 2011-06-30

DNA Ladder Gene Ruler, 100bp plus (Fermentas)

6x Loading Dye (Fermentas)

Method:

1. Production of a 1 % agarose gel

mAgarose = 0.5 g in 50 ml 1x TAE

adding 2 µl gel red

2. Loading samples

5 µl PCR product and 6x loading dye

2 µl DNA ladder gene ruler (1:10)

3. Run

100 V

time: 00:45 h

Results:

lane	Sample	Volume in µl	Expected size in bp
M	marker	2
1	mdnA with SfiI	6	224

125 px

further tasks:

purification of PCR product

purification of PCR product mdnA SfiI for phage display

Investigators: Sabine, Sandrina

Aim:

purification of PCR product mdnA with SfiI restriction sites for phage display (strategy 1)

Time: 2011-06-24, 15:30-16:00

Materials/Methods:

QIAquick Gel Extraction Kit (250)

Further tasks:

digestion of the fragments with SfiI

Agarose gel electrophoresis of myc-tagged mdnA

Investigators: Jessica

Aim: Verification of myc-tagged mdnA

Time: 2011-07-01,13:00-15:30

Materials:

Agarose broad range (Roth)

1x TAE buffer

Gel Red

PCR product: myc-tagged mdnA from 2011-06-29

DNA Ladder Gene Ruler, 100bp plus (Fermentas)

6x Loading Dye (Fermentas)

Method:

1. Production of a 1 % agarose gel

mAgarose = 0.5 g in 50 ml 1x TAE

adding 2 µl gel red

2. Loading samples

45 µl PCR product and 9 µl 6x loading dye

6 µl DNA Ladder Gene Ruler (1:10)

3. Run

100 V

time: 1 h

Results:

lane	Sample	Volume in µl	Expected size in bp
M	marker	6
1	myc-tagged mdnA	27	233
2	myc-tagged mdnA	27	233

Gel excision of bands, stored at -20 °C (PCR box, blue)

Further tasks:

Gel extraction

Restriction digest

29th Labday 2011-07-04

digestion of PCR product mdnA with restriction enzyme SfiI (strategy 1)

Investigators: Sandrina, Sabine

Aim: digestion of mdnA with SfiI for cloning into pak100

Time: 2011-07-04,14:00-16:30

Materials:

Digestion with SfiI

50 µl sample

NEB 10x buffer 4

100x BSA

3 µl restriction enzyme SfiI

PCR product (mdnA with SfiI restriction sites)

Further tasks:
ligation with pak100bla-KDIR

ligation of mdnA into pak100(strategy 1)

Investigators: Sandrina, Sabine

Aim: ligation of mdnA into pak100 for phage display

Time: 2011-07-04,16:00-16:30

Materials:

Ligation: 2 µl vector (pak100 bla KDIR, digested with SfiI), 2 µl insert (mdnA, digested with SfiI), 1 µl quick ligase, 5 µl 2x quick ligase NEB, 10 min, room temperature

Further tasks:
purification

Realization:
amplified mdnA does not contain a myc-tag --> further task: order a new reversed primer for mdnA (strategy 1) containing a myc-tag

Amplification of mdnA for phage display (strategy 2)

Investigators: Sandrina, Sabine

Aim: amplificate mdnA of pARW089 with primers pf_mdnA_sfi_1 and pr_mdnA_sfi_1

Time: 2011-06-10,14:00-16:30

Materials/Methods:

PCR

5 µl Vector pARW089

Eppendorf Mastercycler Gradient (program sabrina)

0,25 µl OneTaq DNA Polymerase (NEB)

1 µl dNTPs

1 µl per primer

31,75 µl DNase free water

10 µl 5xOneTaq Standard Reaction Buffer

Gel electrophoresis PCR product mdnA (strategy 2)

Investigators: Sandrina, Sabine

Aim:verification of mdnA (strategy 2)

Time: 2011-07-04,17:00-19:00

Materials/Methods:

Agarose broad range (Roth)

1x TAE buffer

Gel Red

PCR products: mdnA for phage display (strategy 1

DNA Ladder Gene Ruler, 100bp plus (Fermentas)

6x Loading Dye (Fermentas)

Method:

1. Production of a 1 % agarose gel

mAgarose = 0.5 g in 50 ml 1x TAE

adding 2 µl gel red

2. Loading samples

5 µl PCR product and 6x loading dye

2 µl DNA ladder gene ruler (1:10)

3. Run

100 V

time: 01:15 h

Results:

lane	Sample	Volume in µl	Expected size in bp
1	marker
2	PCR product mdnA	5	ca. 230

150px

30th Labday 2011-07-06

digestion of PCR products mdnA, geneIII and vector pARW089 (strategy 2)

Investigators: Sandrina, Sabine

Aim: digestion of mdnA, gene III and pARW089 for getting an mdnA-geneIII fusion gene with rfc25 restrition sites in pARW089 by ligation of the three fragments

Time: 2011-07-06,14:00-16:00

Materials:

Digestion of mdnA with NarI (EheI isoschizomere) and AgeI

40 µl sample

NEB 10x buffer (4 µl)

1 µl restriction enzyme NarI

1 µ restriction enzyme AgeI

30 µl PCR product (mdnA with rcf 25 and EheI/NarI restriction sites)

4 µl H²O

Digestion of geneIII with NgoMIV and AatII

40 µl sample

NEB 10x buffer (4 µl)

1 µl restriction enzyme NgoMIV

1 µ restriction enzyme AatII

30 µl PCR product (geneIII with rcf 25 and AatII restriction sites)

4 µl H²O

Digestion of vector pARW089 with NarI (EheI isoschizomere) and AatII

20 µl sample

NEB 10x buffer (2 µl)

1 µl restriction enzyme NarI

1 µ restriction enzyme AatII

12 µl vector DNA

4 µl H²O

1 h at 37°C

Further Tasks:

gel electrophoresis and purification of the three digested fragments

Gel extraction from agarose gel of digested fragments mdnA, geneIII and pARW089 (strategy 2)

Investigators: Stefan

Aim: Purification of mdnA, geneIII and pARW089 (strategy 2) to obtain an mdnA-geneIII fusion gene which can be cloned into pARW089

Time: 2011-07-06,17:00-19:00

Materials/Methods:

PCR Clean-up Kit NucleoSpin ExtractII (Machery-Nagel)

Method:

1. Cut out visible DNA bands of the digestion product of a 0.8% and 1% agarose gel, respectivly

2. Use protocol of PCR clean-up

3. Two bands were cut out for the vector and genIII. Just use the lower two bands for first ligation.

Results:

DNA concentration geneIII: 5,4 ng/µl

DNA concentration mdnA: 8,2 ng/µl

DNA concentration pARW089: 3,8 ng/µl

measured by NanoDrop

Further Tasks:

Repeat digestion of pARW089 (because of low concentration)and ligation of the three fragments

31th Labday 2011-07-07

repeated digestion of pARW089 with NarI and AatII (strategy 2)

Investigators: Sabine, Sandrina

Aim: Digestion of pARW089 (strategy 2) so that an mdnA-geneIII fusion gene can be cloned into pARW089

Time: 2011-07-07,10:00-12:00

Materials/Methods:

Digestion of vector pARW089 with NarI (EheI isoschizomere) and AatII

20 µl sample

NEB 10x buffer (2 µl)

1 µl restriction enzyme NarI

1 µ restriction enzyme AatII

3 µl vector DNA (ca. 800 ng/ µl)

13 µl H²O

2 1/2 h at 37°C

afterwards: inactivation of restriction enzymes: 20 min, 65°C

Further tasks:

gel electrophoresis of digested vector

Gel electrophoresis of digested pARW089 vector(strategy 2)

Investigators: Sandrina, Sabine

Aim:Purification of pARW089 (strategy 2) so that an mdnA-geneIII fusion gene can be cloned into pARW089

Time: 2011-07-07,14:30-15:30

Materials/Methods:

Agarose broad range (Roth)

1x TAE buffer

Gel Red

digested vector pARW089

DNA Ladder Gene Ruler 1kb plus(Fermentas)

6x Loading Dye (Fermentas)

Method:

1. Production of a 0,5 % agarose gel

mAgarose = 0.25 g in 50 ml 1x TAE

adding 2 µl gel red

2. Loading samples

digestion product and 6x loading dye

pARW089 digestion product (first try, conc: 3,8 ng/µl) to verify measured concentrations

3. Run

100 V

time: 00:45 h

Results:

lane	Sample	Volume in µl	Expected size in bp
1	marker
2	purificated, digested vetor try 1	30	ca. 10 000
3	digested pARW089 vector	30	ca. 10.000

File:UP versuch2 pARW Verdau.7.7.png

one band was cutted out of the gel for purifacation

Further tasks:

ligation of mdnA, geneIII and pARW089

ligation of mdnA and geneIII into pARW089(strategy 2)

Investigators: Sabine

Aim: ligation of mdnA, geneIII, and pARW089 to get an mdnA-geneIII fusion gene in pARW089for phage display

Time: 2011-07-07,15:30-17:30

Materials:

Ligation: 2 µl vector (pARW089 digested with NarI and AatII), 3 µl insert (mdnA digested with NarI and AgeI, and geneIII digested with AatII and NgoMIV), 1 µl T4 ligase, 1 µl T4 ligase NEB-buffer,over night, 16°C

Further tasks:
transformation of E. coli cells(XL1-blue)

32th Labday 2011-07-12

Transformation of ligation mdnA, geneIII, and pARW089 (2011-07-07) for phage display (strategy 2)

Investigators: Sandrina, Sabine

Aim:amplification of previous ligation

Time: 9:00-11:30

Method:

ligation-samples from 2011-07-07;

(ligation of mdnA, geneIII and pARW089)

protocol:

addition of 10 µl ligation reaction to cells (XL1-blue, tet-resistance) in 1.5 ml Eppi,

incubation 25 min on ice,

heat shock 45 sec at 42°C,

incubation 2 min on ice,

addition of 750 µl LB medium,

incubation at 37 °C for 60 min in Eppendorf thermomixer at 750 rpm,

plating on LB medium with 1,5 % agar, 100 µg/ml ampicillin, 100 µg/µl tetracyclin

storage over night at 37°C

Further tasks:
control cell clones

33th Labday 2011-07-13

Error prone PCR (varied MnCl2 concentration) of mdnA

Investigators: Steffi, Jessica, Katharina

Time: 2011-06-29,14:00-16:00

Aim: Random mutation of mdnA gene based on increased MnCl2 concentration

Materials and Methods: see 26th Labday

34th Labday 2011-07-14

Agarose gel electrophoresis of error prone PCR of mdnA from 2011-07-13 and gel extraction

Investigators: Katharina

Time: 2011-06-29,8:00-11:00

Materials:

Agarose broad range (Roth)

1x TAE buffer Gel Red

10 PCR products of mutated mdnA

DNA Ladder GeneRuler, 100bp plus (1:10) (Fermentas)

6x Loading Dye (Fermentas)

Method:

1. Production of two 1 % agarose gel

For each gel: mAgarose = 0.5 g in 50 ml 1x TAE adding 2 µl gel red

2. Loading samples

50 µl PCR product and 6x loading dye

6 µl DNA Ladder GeneRuler (1:10) (Fermentas)

3. Run

110 V time: 1 h

Results:

	gel 1			gel 2
lane	Sample	Volume in µl	Expected size in bp	Sample	Volume in µl	Expected size in bp
1	marker	6		marker	6
2	PCR sample 1a	50	276	PCR sample 1b	50	276
3	PCR sample 2a	50	276	PCR sample 2b	50	276
4	PCR sample 3a	50	276	PCR sample 3b	50	276
5	PCR sample 4a	50	276	PCR sample 4b	36	276
6	PCR sample 5a	50	276	PCR sample 5b	50	276

300px 300px

The bands were excised and purified using the NucleoSpin Extract II (Macherey-Nagel) extraction Kit.

Further Tasks:

digestion of the purified samples

ligation

Digestion of error prone PCR of mdnA from 2011-07-13 and pARW089 and agarose gel electrophoresis

Investigators: Jessica

Time: 2011-06-29,11:00-15:00

Materials:

Restriction enzymes: AatII, NarI

NEB Buffer 4

PCR products of random mutated mdnA(1a-5a), purified from gel

vector pARW089

Agarose broad range (Roth)

1x TAE buffer

Gel Red

GeneRuler DNA Ladder Mix (1:10) (Fermentas)

GeneRuler 1kb DNA Ladder (Fermentas)

6x Loading Dye (Fermentas)

Method:

1. Digestion:

components	volume of PCR products /µl	volume of pARW089 /µl
DNA	30	10
NEB Buffer 4(10x)	4	4
Enzyme AatII	0.5	0.8
Enzyme NarI	2	2
H₂O	3.5	23.2
Total volume	40	40

digestion at 37°C for 1 h

2. Production of agarose gel

1.7% gel for PCR products: m_Agarose = 0.85 g in 50 ml 1x TAE adding 2 µl gel red

0.8% gel for vector: m_Agarose = 0.4 g in 50 ml 1x TAE adding 2 µl gel red

3. Loading samples

40 µl digest and 8 µl 6x loading dye

4. Run

80 V, time: >1 h

Results:

Gel 1

lane	Sample	Volume in µl
M	GeneRuler DNA Ladder Mix (1:10)	6
1	digested mdnA 1a	48
2	digested mdnA 2a	48
3	digested mdnA 3a	48
4	digested mdnA 4a	48
5	digested mdnA 5a	48

Gel 2

lane	Sample	Volume in µl	Expected size in bp
M	GeneRuler 1kb DNA Ladder	2
1	digested pARW089	48

Gel excision and storage at -20°C (DNA box, orange)

Overnight culture of PDV089

Investigators: Sandrina, Sabine

Aim:
control plasmid ligation (pARW089, mdnA, geneIII --> PDV089, strategy 2)

Time: 2011-07-13,12:00-13:00

Materials/Methods:

LB-medium with tet and amp

cell clones from over night plate

incubate over night at 37°C and 750 rpm

Further tasks:

plasmid preparation and analytic digestion

Repeated PCR of mdnA for phage display (strategy 1) repeated with new ordered reversed primer (with myc-tag) and geneIII

Investigators: Sandrina, Sabine

Time: 2011-06-30,13:00-16:00

Aim:

amplification of mdnA with SfiI restriction sites and myc (vector pARW089) to clone it into pAk100 bla KDIR

amplificate GeneIII of pak100 with primers pf_iGEM_GenIII_NgoMIV and pr_GenIII_iGEM_AatII (phage display strategy 2), because there were two bands after digestion with NgoMIV and AatII after gel electrophoresis

Materials/Methods:

see 2011-06-10

changes:

program: 123, Thermo Hybrid, PX2

further tasks:

gel electrophoresis with PCR product

35th Labday 2011-07-15

Ligation of mutated mdnA into pARW089

Investigators: Katharina

Aim: create pARW089 vector with inserted mutated mdnA

Time: 10:00-14:00

Materials:

Ligation: 2 µl vector (pARW089 digested with NarI and AatII), 3 µl insert (mdnA digested with NarI and AatII), 1 µl T4 ligase, 1 µl T4 ligase NEB-buffer, 4 h at room temperature

Further tasks:
transformation of E. coli cells(XL1-blue)

Transformation of competent E. coli cells with mutated mdnA in pARW089

Investigators: Katharina

Aim:Transformation of ligations

Time: 14:00-15:00

Method:

protocol:

addition of 2 µl ligation reaction to cells (XL1-blue) in 1.5 ml Eppi,

incubation 25 min on ice,

heat shock 45 sec at 42°C,

incubation 5 min on ice,

addition of 750 µl LB medium,

incubation at 37 °C for 60 min in Eppendorf thermomixer at 750 rpm,

plating on LB medium with 1,5 % agar, 100 µg/ml kanamycin

storage over night at 37°C

Further tasks:
control cell clones

ligation of mdnA into pak100(strategy 1)

Investigators: Sandrina, Sabine

Aim: ligation of mdnA into pak100 for phage display

Time: 2011-07-15,11:00-13:00

Materials:

Ligation: 2 µl vector (pak100 bla KDIR, digested with SfiI), 2 µl insert (mdnA, digested with SfiI), 1 µl T4 ligase, 5 µl 2x 10x T4 ligase buffer, 20 min, room temperature

Further tasks:
transformation into competent cells

Transformation of ligation mdnA into PAK100 bla KDIR for phage display (strategy 1)

Investigators: Sandrina, Sabine

Aim:amplification of previous ligation

Time: 13.00-15:00

Method:

ligation-samples

(ligation of mdnA into PAK100 bla KDIR)

protocol:

addition of 10 µl ligation reaction to cells (XL1-blue, tet-resistance) in 1.5 ml Eppi,

incubation 25 min on ice,

heat shock 45 sec at 42°C,

incubation 2 min on ice,

addition of 750 µl LB medium,

incubation at 37 °C for 60 min in Eppendorf thermomixer at 750 rpm,

plating on LB medium with 1,5 % agar, 100 µg/ml ampicillin, 100 µg/µl tetracyclin

storage over night at 37°C

Further tasks:
control cell clones

ligation of mdnA and geneIII for strategy 2

Investigators: Sabine, Sandrina

Aim:ligation of mdnA and geneIII to get an mdnA-geneIII fusion gene phage display

Time: 2011-07-15,15:00-16:00

Materials:

Ligation: 3 µl insert mdnA digested with NarI and AgeI and geneIII digested with AatII and NgoMIV), 1 µl T4 ligase, 1 µl T4 ligase NEB-buffer, 2 µl H2O , 20 min, room temperature

Results:

lane	Sample	Volume in µl	Expected size in bp
M	marker, DNA ladder mix ladder mix	10
1	mdnA ligated with geneIII		ca. 600

500 px

Further tasks:
ligation with pARW089

ligation of the fusion gene mdnA-geneIII into pARW089(strategy 2)

Investigators: Sabine, Sandrina

Aim:ligation of mdnA-geneIII in pARW089 to get an mdnA-geneIII fusion gene in pARW089for phage display

Time: 2011-07-07,17:00-19:00

Materials:

Ligation: 2 µl vector (pARW089 digested with NarI and AatII), 3 µl insert, 1 µl T4 ligase, 1 µl T4 ligase NEB-buffer,over night, 16°C

two bands were observed after mdnA-geneIII ligation (red boxes)--> ligation with pARW089 was tried with both bands

Further tasks:
transformation of E. coli cells(XL1-blue)

36th Labday 2011-07-16

Transformation of ligated pARW089 with mdnA-geneIII

Investigators: Stefan

Aim:Transformation of phage display ligations (pARW089 with mdnA-geneIII) using heat shock

Time: 12:00-20:00

Method/Materials:

1) Take chemically competent E. coli XL1 blu cells from –80°C freezer.

2) Turn on heat block to 42°C.

3) use 60 µL of competent cells for each approach

4) Keep tubes on ice.

5) Add 2 µL DNA solution into the E. coli cell suspension, mix by flicking the tube. Incubate on ice for 15-30 min.

6) Put tubes into heat block at 42°C for 45 seconds.

7) Put tubes back on ice for 2 minutes to reduce damage to the E. coli cells.

8) Add 750 µL of LB with no antibiotic. Incubate tubes for 1 hour at 37°C and 750 rpm.

9) spin down the cell suspension for 3 min at 6000 rcf at 4 °C , discard most of the supernatant, resuspend the bacterial pellet by pipetting and use this for spreading.

10) Spread 100 ul of the resulting culture on LB agar plates with Tet/CM.

11) Grow overnight at 37 °C.

12) Pick colonies about 12-16 hours later.

Further tasks:
picking clones and plasmid preparation

Overnight culture of XL1 blue with mdnA in PAK100blaKDIR

Investigators: Stefan

Aim: Picking clone 1 from plate and do an overnight culture

Time: 12:00-20:00

Method/Materials:

Clone ID_1: Strain:XL1 blue, Plasmid:mdnA in PAR100bla KDIR, Clone number:1

15 mL of LB media were supplied with 15 µL of Tet and CM and cells were incubated at 37 °C overnight.

Further tasks:
plasmid preparation

37th Labday 2011-07-17

Miniprep of E. coli overnight culture containing PAK100blaKDIR

Investigators: Stefan

Aim: isolate the plasmid

Time: 12:00-20:00

Method/Materials:

protocol 5.1 of the NucleoSpin Plasmid Kit was used

Further tasks:
sequence the plasmid

E. coli XL1 blue stock culture containing PAK100blaKDIR with mdnA (Phage Display-strategy 1)

Investigators: Stefan

Aim: set up a stock culture

Time: 12:00-20:00

Method/Materials:

2 x 700 µL culture were mixed with 300 µL of glycerol and stored in -80 °C freezer

Further tasks:

overnight culture of E. coli Xl blue m. Ligation089jessi, u.Ligationvector089jessi,u.Ligationu.BandemdnAgeneIIIpARW089, mLigatiojnm.BandemdnAgeneIIIpARW089

Investigators: Stefan

Aim: pick two clones each and inoculate them overnight in 15 mL LB supplied with 15 µL Tet and Amp

Time: 12:00-20:00

Method/Materials:

Further tasks:
plasmid preparation

38th Labday 2011-07-18

overnight culture of E. coli XL-1 blue containing mutated mndA in parW089

Investigators: Katharina

Aim: Picking 5 clones from each plate and do an overnight culture

Time: 14:00-15:00

Method/Materials:

E. coli Strain:XL1 blue, Plasmid:mdnA in parW089

5 mL of LB media were supplied with 5 µL of Kan and cells were incubated at 37 °C overnight.

Further tasks:
plasmid preparation

Dual-Tranformation of XL1-blu (E. coli) with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla

Investigators: Sascha

Aim:

Dual-Transformation of XL1-blue to test the in-vivo selection system (pTEV-SCS1 + pJC354_ssTorA_NheI_CS-TEV_XhoI_bla)

Materials:

Plasmids:

- pTEV-SCS1 (TEV protease, res. against kanamycin)

- pJC354_ssTorA_NheI_CS-TEV_XhoI_bla (res. against chloramphenicol)

competent cells XL1-blue

LB media and diffenet antibiotics

Used method:

standard protocol for heat shock transformation

Output: transformed cultures with plasmids

XL1-blue without plasmid (negativ control - no growth over night)

XL1-blue transformed with pTEV-SCS1 - growth with kanamycin in LB media

XL1-blue transformed with pJC354_ssTorA_NheI_CS-TEV_XhoI_bla - growth with chloramphenicol in LB media

XL1-blue transformed with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla - growth with kanamycin and chloramphenicol in LB media

Further tasks:

reinfect media with cultures for new growth period and distribution to LB Agar with different antibiotic concentrations

Test digestion of ligations for strategies 1 and 2

Investigators: Sandrina

Aim:control if liagation of geneIII and mdnA into pARW089 (strategy 2) and ligation of mdnA into PAK100 bla KDIR worked

Time: 15:30-19:00

Materials/Methods:

Strategy 1:

0,5 µl SfiI

2 µl 10x buffer 4 (NEB)

0,2 µl BSA

10 µl vector DNA

13,3 µl H2O

incubate for 1 h at 50°C

Strategy 2:

0,5 µl XbaI

0,5 µl SpeI

2 µl 10x buffer 4 (NEB)

0,2 µl BSA

10 µl vector DNA

12,8 µl H2O

incubate for 1 h at 37°C

Results:

expected bands: strategy 1: ca. 5000 bp and 200bp

expected bands: strategy 2: ca. 5000 bp, 4000 bp, 600 bp and 200

observed bands:

srtategy 1: ca. 5000 and 800 bp

strategy 2: ca. 5000 and 200 bp

Further tasks:

repeat ligations

39th Labday 2011-07-19

Resistance test of the Dual-Transformation of XL1-blue (E. coli) with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla

Investigators: Stefan and Sebastian

Aim:

Dual-Transformation of XL1-blue to test the in-vivo selection system (pTEV-SCS1 + pJC354_ssTorA_NheI_CS-TEV_XhoI_bla)

Materials:

Overnight cultures

- XL1-blue transformed with pTEV-SCS1 - growth with kanamycin in LB media

- XL1-blue transformed with pJC354_ssTorA_NheI_CS-TEV_XhoI_bla - growth with chloramphenicol in LB media

- XL1-blue transformed with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla - growth with kanamycin and chloramphenicol in LB media

LB media and different antibiotics

Used method:

growth over night

set OD (600 nm) after measurement cells were dilutes to OD 0,1, transformed XL1-blue grow again to OD 0,5. Out of this approach a dilution to OD= 0.002 was done and platted to LB agar with kanamycin, IPTG and different concentrations of ampicillin (range form 0-800 ng/ml ampicillin)

Output:

survival of dual transformed cells...

Further tasks:

There was no growth on the TorAbla plates due to the choice of the wrong antibiotic. This was set up again as well as well as the double transformed cells.

Stock cultures of XL1-blue(E. coli)with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla and of XL1-blue(E. coli)with pTEV-SCS1 and of XL1-blue(E. coli)pJC354_ssTorA_NheI_CS-TEV_XhoI_bla

Investigators: Stefan

Aim:

make stock cultures

Materials:

Overnight cultures were used. 700 µL culture were spun down to remove antibiotics. The pellets is resuspended in 700 µL LB media and 300 µL glycerol.

Used method:

Further tasks:

Design of Primers for BioBrick mdnA and mdn-Cluster

Investigators: Nicole, Nadine

Aim: Design primers for mdnA and mdn-Cluster(from pARW089) for BioBricks pBAD_mdn_Cluster, pSB_mdn_Cluster and pSB_mdnA

Time: 2011-07-19,14:00-17:00

Materials:

Geneious Pro 5.1.7

Method:

Primer design

- melting temperature based on 4+2 method (for mdnA-overlapping part)

- adding sequences for restriction sites (considering reading frame)

- Check self-complementarity and melting temperature using Oligo Calc

Plan:

mdn-Cluster BioBricks

mdnA BioBrick

Results:

Primer sequences

600px

Further tasks:

do PCR of mdnA

Miniprep of E. coli Xl blue containing mutated mndA in parW089

Investigators: Niels

Aim: 25x overnight cultures :

- miniprep

Time: 2011-07-19,12:00-16:00

Materials:

E. coli Xl blue

Kit:

Nucleic Acid and Protein Purification

- Support protocol for NucleoSpin Plasmid

Results:

Nanodrop:

Further task:

sequencing

digest

40th Labday 2011-07-20

Resistance test of the Dual-Transformation of XL1-blue (E. coli) with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla

Investigators: Stefan

Aim:

overnight cultures for resistance test

Materials:

Overnight cultures of

- XL1-blue transformed with pJC354_ssTorA_NheI_CS-TEV_XhoI_bla - growth with chloramphenicol in LB media

- XL1-blue transformed with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla - growth with kanamycin and chloramphenicol in LB media

Used method:

Restriction enzyme digestion pARW089 and pARW071

Investigators: Katharina, Nadine

Time: 2011-07-21,11:00-12:00

Aim: Restriction enzyme digestion of pARW089 and pARW071

Materials:

Restriction enzymes: AatII, EheI (both from Elke)

NEB Buffer 4

Plasmids: pARW089, pARW071

Problem:

AatII was empty (ordered new batch for tomorrow)

overnight culture of E. coli Xl blue m. ligation mdnA and geneIII into pARW089

Investigators: Sandrina

Aim: repeated test digestion of ligation mdnA and geneIII into pARW089 from 2011-07-12/13

Time: 16:30-18:00

Method/Materials:

LB medium

Further tasks:
plasmid preparation and test digestion

Repeated PCR of mdnA and gene III for phage display (strategy 1+2)

Investigator: Sandrina

Time: 2011-07-26,9:00-11:00

Aim:

amplification of mdnA with SfiI restriction sites (strategy 1)

amplification of mdnA with NarI and AgeI and rfc 25 restriction sites (strategy 2)

amplificate GeneIII with NgoMIV and AatII and rfc 25 restriction sites (strategy 2)

Primer:

primer: pf_sfi-mdnA_2 and pr_sfi_mdnA_myc (mdnA, strategy 1)

primer: pf_mdnA_iGEM_EheI and pr_mdnA_iGEM_AatII (mdnaA, strategy 2)

primer: pf_geneIII_NgoMIV and pr_geneIII_iGEM_AatII (geneIII, strategy 2)

Reaction Components:

5 µl Vector pARW089

0,25 µl OneTaq Polymerase

1 µl dNTPs

1 µl per primer

5 µl 5x PCR Buffer

37,75 µl DNase free water

purification of PCR fragments with QIAquick Gel Extraction Kit (250)

Further tasks:

digestion of PCR products

digestion of PCR products and vectors (strategy 1+2)

Investigator: Sandrina

Time: 2011-07-26,11:00-13:00

Aim:

digestion of mdnA and gene III for getting an mdnA-geneIII fusion gene with rfc25 restrition sites after ligation

digestion of pARW089 for ligation of mdnA/geneIII fusion gene into it (strategy 2)

digestion of pak100blaKDIR and and mdnA to enable ligation

Time: 2011-07-26,14:00-17:30

digestion enzymes:

digestion of mdnA (strategy 2) with NarI and AgeI

digestion of geneIII (strategy 2) with NgoMIV and AatII

digestion of pARW089 (strategy 2) with NarI and AatII

digestion of pak100blaKDIR (strategy 1) with SfiI

digestion of mdnA (strategy 1) with SfiI

reaction components:

4 µl NEB 10x buffer

1 µl per restriction enzyme

30 µl PCR product / 5 µl vector

4 µl H²O

BSA (only Sfi digestion)

reaction conditions:

1 h for PCR fragments

3 h for plasmids

50°C (SfiI digestion)

37°C NarI, AgeI, NgoMIV and AatII digestion

ligation of mdnA and geneIII into digested pARW089(strategy 2)

Investigators: Sandrina

Aim: get phage display vector pPDV089

Time: 2011-07-20,14:00-16:00

Materials:

6 µl digested vector pARW089 (NarI, AatII)

1 µl geneIII

1 µl mdnA

vector and insert should be added in ratio 1:3

2 µl 10x T4 Ligase Buffer

1 µl T4 Ligase

9 µl water

incubation over night at 16°C

Further Tasks: transormation of competent cells

ligation of mdnA into pak100blaKDIR (stategy 1)

Investigator: Sandrina

Aim: generate phage display vector pPDV100 (strategy 1)

Time: 2011-07-20,14:00-16:00

Method/Materials:

3 µl (ca 90 ng) Sfi-digested vector pak100

1 µl (ca 20 ng) Sfi-digested PCR fragment mdnA

2 µl 10x T4 Ligase Buffer

1 µl T4 Ligase

13 µl water

incubate over night at 16°C

Further Tasks: transformation of competent cells

41th Labday 2011-07-21

Test digestion of ligation for strategy 2

Investigators: Sandrina

Aim:repeated test digestion of ligation mdnA and geneIII into pARW089 from 2011-07-12/13

Time: 15:30-19:00

Materials/Methods:

Strategy 2:

0,5 µl XbaI

0,5 µl SpeI

2 µl 10x buffer 4 (NEB)

0,2 µl BSA

10 µl vector DNA

12,8 µl H2O

incubate for 1 h at 37°C

Results:

lane	Sample	Volume in µl	Expected size in bp
M	marker, DNA ladder mix, fermentas	2
1-20	digested ligations	6	ca. 5000 bp, 4000 bp, 600 bp and 200

400 px

Further tasks:

sequencing of clone 6

Transformation of ligated pARW089 with mdnA-geneIII and ligated PAK100 bla KDIR with mdna

Investigators: Sandrina

Aim:Transformation of phage display ligations (strategy 1 and2) (2011-7-20) using heat shock

Time: 10:00-12:00

Method/Materials:

1) Take chemically competent E. coli XL1 blu cells from –80°C freezer.

2) Turn on heat block to 42°C.

3) use 60 µL of competent cells for each approach

4) Keep tubes on ice.

5) Add 2 µL DNA solution into the E. coli cell suspension, mix by flicking the tube. Incubate on ice for 15-30 min.

6) Put tubes into heat block at 42°C for 45 seconds.

7) Put tubes back on ice for 2 minutes to reduce damage to the E. coli cells.

8) Add 750 µL of LB with no antibiotic. Incubate tubes for 1 hour at 37°C and 750 rpm.

9) spin down the cell suspension for 3 min at 6000 rcf at 4 °C , discard most of the supernatant, resuspend the bacterial pellet by pipetting and use this for spreading.

10) Spread 100 ul of the resulting culture on LB agar plates with Tet/CM.

11) Grow overnight at 37 °C.

12) Pick colonies about 12-16 hours later.

Further tasks:
picking clones and plasmid preparation

42th Labday 2011-07-22

Restriction enzyme digestion pARW089 and pARW071 AND trouble-shouting

Investigators: Katharina, Nadine

Time: 2011-07-22,10:00-15:00

Aim: Restriction enzyme digestion of pARW089 and pARW071

Materials:

Restriction enzymes: AatII, NarI

NEB Buffer 4

Plasmids: pARW089, pARW071

43th Labday 2011-07-23

Restriction enzyme digestion pARW089 (different incubation times), plasmid purification and ligation with EP-PCR mdnA

Investigators: Nadine

Time: 2011-07-22,14:30-20:30

Aim: Restriction enzyme digestion of pARW089 with different incubation times to test best digestion conditions, problems with the digestion of pARW089 in the last trials (-->gel excision and ligation if it worked out

Materials:

Restriction enzymes: AatII, NarI, control: XmaI

NEB Buffer 4

PCR products of random mutated mdnA(1a-5a), purified from gel

vector pARW089

Agarose broad range (Roth)

1x TAE buffer

Gel Red

GeneRuler 1kb DNA Ladder (Fermentas)

6x Loading Dye (Fermentas)

Method:

1. Digestion:

components	volume of pARW089 /µl
DNA	5
NEB Buffer 4(10x)	4
Enzyme AatII	0.8
Enzyme NarI	2
H₂O	28.2
Total volume	40

2. Digestion: control

components	volume of pARW089 /µl
DNA	5
NEB Buffer 4(10x)	4
Enzyme XmaI	1
BSA	0.2
H₂O	29.2
Total volume	40

digestion at 37°C for 1 h

3. Production of agarose gel

0.8% gel for vector: m_Agarose = 0.4 g in 50 ml 1x TAE adding 4 µl gel red

4. Loading samples

40 µl digest and 8 µl 6x loading dye

5. Run

110 V, time: >40 min

Results:

Gel

lane	Sample	Volume in µl	Expected size in bp
M	GeneRuler 1kb DNA Ladder	2	10296, 116
1	digested pARW089 1 hr	48	10296, 116
2	digested pARW089 2 hrs	48	10296, 116
3	digested pARW089 2.5 hrs	48	10296, 116
4	digested pARW089 XmaI 3 hrs	48	3270, 7142
5	digested pARW089 3 hrs	48	10296, 116
6	digested pARW089 undigested	48

250px

6. Gel excision

lane 4 and 6, band: 10296, respectively

7. Ligation

Ligation: 3 µl vector (pARW089 digested with NarI and AatII, incubation 2.5 hrs and 3 hrs), 3 µl insert (mdnA digested with NarI and AatII from 2011-07-29), 1 µl T4 ligase, 1 µl T4 ligase NEB-buffer, overnight at 16°C

samples:

pARW089 (dig. 2.5 hrs) + mdnA 1A

pARW089 (dig. 2.5 hrs) + mdnA 2A

pARW089 (dig. 2.5 hrs) + mdnA 3A

pARW089 (dig. 2.5 hrs) + mdnA 4A

pARW089 (dig. 2.5 hrs) + mdnA 5A

pARW089 (dig. 3 hrs) + mdnA 1A

pARW089 (dig. 3 hrs) + mdnA 2A

pARW089 (dig. 3 hrs) + mdnA 3A

pARW089 (dig. 3 hrs) + mdnA 4A

pARW089 (dig. 3 hrs) + mdnA 5A

Further tasks:
transformation of E. coli cells(XL1-blue)

creation of pPDV089 in Genious

Investigators: Sandrina, Sabine

Aim: getting a model of pPDV089 for sequence alignment with purified pPDV089 plasmids from clones

Time: 09.00-11.00

Method/Materials:

software Genious

Further tasks:

perform alignment

sending pPDV089 from positive clone to GATC for sequencing

Investigators: Sabine

Aim: get sequence of generated phage display vector pPDV089 (strategy 2) to control the ligation of digested pARW089 with digested mdnA and gene III

Method/Materials:

10 µl of purified pPDV089 and 20 µl primer sf_mdna_1 were sent to GATC

Further tasks:

perform alignment

44th Labday 2011-07-24

Transform ligation from 42th Labday into E. coli XL blue 1 cells

Investigators: Stefan

Time: 2011-07-22,11:00-15:00

Aim: cells containing the desired vectors

Materials/Methods:

1)Take chemically competent E. coli XL1 blu cells from –80°C freezer.

2) Turn on heat block to 42°C.

3) use 60 µL of competent cells for each approach

4) Keep tubes on ice.

5) Add 2 µL DNA solution into the E. coli cell suspension, mix by flicking the tube. Incubate on ice for 15-30 min.

6) Put tubes into heat block at 42°C for 45 seconds.

7) Put tubes back on ice for 2 minutes to reduce damage to the E. coli cells.

8) Add 750 µL of LB with no antibiotic. Incubate tubes for 1 hour at 37°C and 750 rpm.

9) spin down the cell suspension for 3 min at 6000 rcf at 4 °C , discard most of the supernatant, resuspend the bacterial pellet by pipetting and use this for spreading.

10) Spread 100 ul of the resulting culture on LB agar plates with Tet/CM. 11) Grow overnight at 37 °C. 12) Pick colonies about 12-16 hours later.

45th Labday 2011-07-25

overnight culture of E. coli XL-1 blue containing mutated mndA in parW089

Investigators: Steffi

Aim: Picking 1 clone from each plate (Ligation pARW089 mdnA 2hrs 1A-5A, Ligation pARW089 mdnA 3hrs 1A-5A) and do an overnight culture

Time: 16:00-17:00

Method/Materials:

E. coli Strain: XL1 blue, Plasmid:mdnA in parW089

5 mL of LB media were supplied with 5 µL of Kan and cells were incubated at 37 °C overnight.

Further tasks:
plasmid preparation

46th Labday 2011-07-26

Miniprep of E. coli Xl blue containing mutated mndA in parW089

Investigators: Steffi

Aim: 10x overnight cultures :

- miniprep

Time: 2011-07-26,09:00-16:00

Materials:

E. coli Xl blue

Kit:

Nucleic Acid and Protein Purification

- Support protocol for NucleoSpin Plasmid

Further task:

sequencing

Alignment of sequenced pPDV089 and model of pPDV089

Investigator: Sabine

Time: 2011-07-26,10:30-11:30

Aim: control of generated phage display vector pPDV089

Materials/Methods:

file sent from GATC

software Genious

Results:

2 mutations in geneIII

the last 2 aminoacids of myc are absent

Further task:

repeat PCR of all fragments using polymerase S (not OneTaq as before)

Repeated PCR of mdnA and gene III for phage display (strategy 1+2)

Investigator: Sabine

Time: 2011-07-26,11:30-13:30

Aim:

amplification of mdnA with SfiI restriction sites (strategy 1)

amplification of mdnA with NarI and AgeI restriction sites (strategy 2)

amplificate GeneIII with NgoMIV and AatII restriction sites (strategy 2)

Primer:

primer: pf_sfi-mdnA_2 and pr_sfi_mdnA_myc (mdnA, strategy 1)

primer: pf_mdnA_iGEM_EheI and pr_mdnA_iGEM_AatII (mdnaA, strategy 2)

primer: pf_geneIII_NgoMIV and pr_geneIII_iGEM_AatII (geneIII, strategy 2)

Reaction Components:

5 µl Vector pARW089

0,25 µl Taq Polymerase S (BioScience)

1 µl dNTPs

1 µl per primer

5 µl 10x PCR Buffer S

37,75 µl DNase free water

purification of PCR fragments with QIAquick Gel Extraction Kit (250)

Further tasks:

digestion

digestion of PCR products and vectors (strategy 1+2)

Investigator: Sabine

Aim:

digestion of mdnA and gene III for getting an mdnA-geneIII fusion gene with rfc25 restrition sites after ligation

digestion of pARW089 for ligation of mdnA/geneIII fusion gene into it (strategy 2)

digestion of pak100blaKDIR and and mdnA to enable ligation

Time: 2011-07-26,14:00-17:30

digestion enzymes:

digestion of mdnA (strategy 2) with NarI and AgeI

digestion of geneIII (strategy 2) with NgoMIV and AatII

digestion of pARW089 (strategy 2) with NarI and AatII

digestion of pak100blaKDIR (strategy 1) with SfiI

digestion of mdnA (strategy 1) with SfiI

reaction components:

4 µl NEB 10x buffer

1 µl per restriction enzyme

30 µl PCR product / 5 µl vector

4 µl H²O

BSA (only Sfi digestion)

reaction conditions:

1 h for PCR fragments

3 h for plasmids

50°C (SfiI digestion)

37°C NarI, AgeI, NgoMIV and AatII digestion

Further Tasks:

gel electrophoresis for purification of the digested fragments and vectors

gel electrophoresis of digested fragments and vectors

Investigator: Sabine

Aim: control PCR and digestion

Time: 2011-07-06,18:00-19:00

Results:

sizes of bands

pak100blaKDIR (SfiI, strategy 1): ca 4,5 kp

pARW089 (NarI and AatII, strategy 2): ca 10,5 kb

mdnA (SfiI, strategy 1): ca 250 bp

mdnA (NarI and AgeI, stategy 2): ca 200 bp

geneIII (NgoMIV and AatII): no band

purification with QIAquick Gel Extraction Kit (250)

Further Tasks:

repeat PCR of geneIII

ligation

Repeated PCR gene III for phage display (strategy 2)

Investigator: Sabine

Time: 2011-07-26,19:00-19:30

Aim:

amplificate GeneIII with NgoMIV and AatII restriction sites (strategy 2)

Primer:

primer: pf_geneIII_NgoMIV and pr_geneIII_iGEM_AatII (geneIII, strategy 2)

Reaction Components:

5 µl pak100blaKDIR

0,25 µl Taq Polymerase S (BioScience)

1 µl dNTPs

1 µl per primer

5 µl 10x PCR Buffer S

37,75 µl DNase free water

Further tasks:

purification

digestion

47th Labday 2011-07-27

digestion of PCR product geneIII (strategy 2)

Investigator: Sabine

Aim: digestion of gene III for getting an mdnA-geneIII fusion gene with rfc25 restrition sites after ligation

Time: 2011-07-26,09:30-10:30

reaction components:

4 µl NEB 10x buffer

1 µl per restriction enzyme (NgoMIV and AatII

30 µl PCR product

4 µl H²O

reaction conditions:

1 h, 37°C

Further Tasks:

gel electrophoresis for purification of the digested geneIII

gel electrophoresis of digested geneIII

Investigator: Sabine

Aim: control PCR and digestion

Time: 2011-07-27,10:30-11:30

Method/Materials:

1% agarose gel

GeneRuler DNA Ladder Mix (SM0331)

5x loading dye

purification with QIAquick Gel Extraction Kit (250)

Results: size of band: ca 550 bp

Further Tasks: ligation

ligation of mdnA into pak100blaKDIR (stategy 1)

Investigator: Sabine

Aim: generate phage display vector pPDV100 (strategy 1)

Time: 2011-07-27,11:30-12:30

Method/Materials:

3 µl (ca 90 ng) Sfi-digested vector pak100

1 µl (ca 20 ng) Sfi-digested PCR fragment mdnA

2 µl 10x T4 Ligase Buffer

1 µl T4 Ligase

13 µl water

1 h, room temperature

Further Tasks: transormation

Transformation of generated pPDV100 (strategy 1)

Investigators: Sabine

Aim:amplification of pPDV100

Time: 12:30-14:00

Method:

addition of 10 µl ligation reaction to XL1-blue cells

incubation 25 min on ice,

heat shock 45 sec at 42°C,

incubation 2 min on ice,

addition of 750 µl LB medium,

incubation 60 min at 37 °C and 750 rpm

plating on agar plates containing 100 µg/ml chloramphenicol

storage over night at 37°C

Further tasks:
control cell clones

48th Labday 2011-07-28

overnight culture of 10 picked clones of XL blue cells transformed with pPDV100

Investigators: Sabine

Aim: amplification and purification of generated phage display vector pPDV100 for test digestion and sequencing

Time: 10:00-10:30

Method/Materials:

5 ml LB medium per clone containining 100 µg/ml chloramphenicol

storage over night at 37°C and 800 rpm

Further tasks:

plasmid preparation, test digestion and sequencing

ligation of mdnA and geneIII (strategy 2)

Investigators: Sabine

Aim: get mdnA/geneIII fusion gene for cloning into pARW089

Time: 2011-07-28,11:00-12:30

Materials:

5 µl (ca 25 ng) digested geneIII (NgoMIV, AatII)

3 µl (ca 30 ng) digested mdnA (NarI, AgeI)

2 µl 10x T4 Ligase Buffer

1 µl T4 Ligase

9 µl water

1 h, room temperature

Further Tasks: ligation of mdnA/geneIII into digested pARW089

ligation of mdnA/geneIII into digested pARW089(strategy 2)

Investigators: Sabine

Aim: get phage display vector pPDV089

Time: 2011-07-28,12:30-13:00

Materials:

6 µl (ca 70 ng) digested vector pARW089 (NarI, AatII)

1 µl (ca 5 ng) fusion gene mdnA/geneIII

2 µl 10x T4 Ligase Buffer

1 µl T4 Ligase

10 µl water

1 h, room temperature

Further Tasks: transormation

Transformation of generated pPDV089 (strategy 2)

Investigators: Sabine

Aim: amplification of pPDV089

Time: 13:30-15:00

Method:

addition of 10 µl ligation reaction to XL1-blue cells

incubation 25 min on ice,

heat shock 45 sec at 42°C,

incubation 2 min on ice,

addition of 750 µl LB medium,

incubation 60 min at 37 °C and 750 rpm

plating on agar plates containing 100 µg/ml tetracyclin and 100 µg/µl ampicillin

storage over night at 37°C

Further tasks:
control cell clones

49th Labday 2011-07-29

Miniprep of E. coli overnight culture containing pPDV100

Investigators: Sabine

Aim: purification of pPDV100 for test digestion and sequencing

Time: 10:00-11:00

Method/Materials: see protocol 5.1 of the NucleoSpin Plasmid Kit

Further tasks: test digestion

gel electrophoresis of digested pPDV100

Investigator: Sabine

Aim: control ligated pPDV100

Time: 2011-07-29,11:30-12:30

Method/Materials:

1% agarose gel, GeneRuler DNA Ladder Mix (SM0331), 5x loading dye

Results: ligation of mdnA into pak100 has not worked

Further Tasks: control all working steps, repeat cloning

creation of pPDV100 in Genious

Investigators: Sandrina, Sabine

Aim: getting a model of pPDV100 for sequence alignment with purified pPDV089 plasmids from clones

Time: 14.00-16.00

Method/Materials: software Genious

Results: subsequent insertion of myc into reverse PCR primer (mdnA) caused frameshift, new primer ordered

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 {{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}
-<html>
+<h2 style="background-color: rgb(240, 20, 70);">28th Labday 2011-07-01</h2>
-<div id="bodycontent">
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Agarose gel electrophoresis of PCR (mdnA for phage display, strategy 1) </h3>
-<h2>July</h2>
+<b>Investigators:</b> Sabine, Sandrina <br>
-<p>
-Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
-</p>
-</div>
+<b>Aim:</b> Verification of mdnA for phage display (strategy 1)<br>
-</html>
+<b>Time:</b> 2011-06-30,14:00-15:30<br>
+<b>Materials:</b><br>
+* Agarose broad range (Roth)
+* 1x TAE buffer
+* Gel Red
+* PCR products: mdnA for phage display (strategy 1) from 2011-06-30
+* DNA Ladder Gene Ruler, 100bp plus (Fermentas)
+* 6x Loading Dye (Fermentas) <br>
+<b>Method:</b><br>
+<b>1. Production of a 1 % agarose gel</b> <br>
+* m<small>Agarose</small> = 0.5 g in 50 ml 1x TAE
+* adding 2 µl gel red <br>
+<b>2. Loading samples</b>
+* 5 µl PCR product and 6x loading dye
+* 2 µl DNA ladder gene ruler (1:10) <br>
+<b>3. Run</b>
+* 100 V
+* time: 00:45 h <br>
+<b>Results:</b><br>
+{| border="1" class="wikitable"
+! lane !! Sample !! Volume in µl !! Expected size in bp
+|-
+| M || marker || 2 ||
+|-
+| 1 || mdnA with SfiI || 6 || 224
+|-
+|}
+[[File:UP PCR mdnA sfi strategy1.png |125 px]] <br>
+<b>further tasks:</b><br>
+purification of PCR product
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">purification of PCR product mdnA SfiI for phage display</h3>
+<b>Investigators:</b> Sabine, Sandrina<br>
+<b>Aim:</b><br>
+* purification of PCR product mdnA with SfiI restriction sites for phage display (strategy 1)<br>
+<b>Time:</b> 2011-06-24, 15:30-16:00 <br>
+<b>Materials/Methods:</b><br>
+* QIAquick Gel Extraction Kit (250)
+<b>Further tasks:</b><br>
+* digestion of the fragments with SfiI
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">Agarose gel electrophoresis of myc-tagged mdnA</h3>
+<b>Investigators:</b> Jessica<br>
+<b>Aim:</b> Verification of myc-tagged mdnA<br>
+<b>Time:</b> 2011-07-01,13:00-15:30<br>
+<b>Materials:</b><br>
+* Agarose broad range (Roth)
+* 1x TAE buffer
+* Gel Red
+* PCR product: myc-tagged mdnA from 2011-06-29
+* DNA Ladder Gene Ruler, 100bp plus (Fermentas)
+* 6x Loading Dye (Fermentas) <br>
+<b>Method:</b><br>
+<b>1. Production of a 1 % agarose gel</b> <br>
+* m<small>Agarose</small> = 0.5 g in 50 ml 1x TAE
+* adding 2 µl gel red <br>
+<b>2. Loading samples</b>
+* 45 µl PCR product and 9 µl 6x loading dye
+* 6 µl DNA Ladder Gene Ruler (1:10) <br>
+<b>3. Run</b>
+* 100 V
+* time: 1 h <br>
+<b>Results:</b><br>
+{| border="1" class="wikitable"
+! lane !! Sample !! Volume in µl !! Expected size in bp
+|-
+| M || marker || 6 ||
+|-
+| 1 || myc-tagged mdnA || 27 || 233
+|-
+| 2 || myc-tagged mdnA || 27 || 233
+|-
+|}
+[[File: UP_AG_PCR_mdnAmycC_2011-07-01_JE.jpg |125 px]] <br>
+*Gel excision of bands, stored at -20 °C (PCR box, blue)
+<b>Further tasks:</b><br>
+* Gel extraction
+* Restriction digest
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">29th Labday 2011-07-04</h2>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">digestion of PCR product mdnA with restriction enzyme SfiI (strategy 1)</h3>
+<b>Investigators:</b> Sandrina, Sabine<br>
+<b>Aim:</b> digestion of mdnA with SfiI for cloning into pak100<br>
+<b>Time:</b> 2011-07-04,14:00-16:30<br>
+<b>Materials:</b><br>
+Digestion with SfiI
+* 50 µl sample
+* NEB 10x buffer 4
+* 100x BSA
+* 3 µl restriction enzyme SfiI
+* PCR product (mdnA with SfiI restriction sites)
+<b>Further tasks:</b><br> ligation with pak100bla-KDIR
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">ligation of mdnA into pak100(strategy 1)</h3>
+<b>Investigators:</b> Sandrina, Sabine<br>
+<b>Aim:</b> ligation of mdnA into pak100 for phage display<br>
+<b>Time:</b> 2011-07-04,16:00-16:30<br>
+<b>Materials:</b><br>
+Ligation: 2 µl vector (pak100 bla KDIR, digested with SfiI), 2 µl insert (mdnA, digested with SfiI), 1 µl quick ligase, 5 µl 2x quick ligase NEB, 10 min, room temperature<br>
+<b>Further tasks:</b><br> purification <br>
+<b>Realization:</b><br> amplified mdnA does not contain a myc-tag --> further task: order a new reversed primer for mdnA (strategy 1) containing a myc-tag
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Amplification of mdnA for phage display (strategy 2)</h3>
+<b>Investigators:</b> Sandrina, Sabine <br>
+<b>Aim:</b> amplificate mdnA of pARW089 with primers pf_mdnA_sfi_1 and pr_mdnA_sfi_1<br>
+<b>Time:</b> 2011-06-10,14:00-16:30<br>
+<b>Materials/Methods:</b><br>
+PCR<br>
+* 5 µl Vector pARW089
+* Eppendorf Mastercycler Gradient (program sabrina)
+* 0,25 µl OneTaq DNA Polymerase (NEB)
+* 1 µl dNTPs
+* 1 µl per primer
+* 31,75 µl DNase free water
+* 10 µl 5xOneTaq Standard Reaction Buffer
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Gel electrophoresis PCR product mdnA (strategy 2)</h3>
+<b>Investigators:</b> Sandrina, Sabine <br>
+<b>Aim:</b>verification of mdnA (strategy 2) <br>
+<b>Time:</b> 2011-07-04,17:00-19:00<br>
+<b>Materials/Methods:</b><br>
+* Agarose broad range (Roth)
+* 1x TAE buffer
+* Gel Red
+* PCR products: mdnA for phage display (strategy 1
+* DNA Ladder Gene Ruler, 100bp plus (Fermentas)
+* 6x Loading Dye (Fermentas) <br>
+<b>Method:</b><br>
+<b>1. Production of a 1 % agarose gel</b> <br>
+* m<small>Agarose</small> = 0.5 g in 50 ml 1x TAE
+* adding 2 µl gel red <br>
+<b>2. Loading samples</b>
+* 5 µl PCR product and 6x loading dye
+* 2 µl DNA ladder gene ruler (1:10) <br>
+<b>3. Run</b>
+* 100 V
+* time: 01:15 h <br>
+<b>Results:</b><br>
+{| border="1" class="wikitable"
+! lane !! Sample !! Volume in µl !! Expected size in bp
+|-
+| 1 || marker || ||
+|-
+| 2 || PCR product mdnA || 5 || ca. 230
+|-
+|}
+[[File: MdnA_strtegy_2, new primer.png|150px]] <br>
+<h2 style="background-color: rgb(240, 20, 70);">30th Labday 2011-07-06</h2>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">digestion of PCR products mdnA, geneIII and vector pARW089 (strategy 2)</h3>
+<b>Investigators:</b> Sandrina, Sabine<br>
+<b>Aim:</b> digestion of mdnA, gene III and pARW089 for getting an mdnA-geneIII fusion gene with rfc25 restrition sites in pARW089 by ligation of the three fragments<br>
+<b>Time:</b> 2011-07-06,14:00-16:00<br>
+<b>Materials:</b><br>
+Digestion of mdnA with NarI (EheI isoschizomere) and AgeI
+* 40 µl sample
+* NEB 10x buffer (4 µl)
+* 1 µl restriction enzyme NarI
+* 1 µ restriction enzyme AgeI
+* 30 µl PCR product (mdnA with rcf 25 and EheI/NarI restriction sites)
+* 4 µl H²O
+Digestion of geneIII with NgoMIV and AatII
+* 40 µl sample
+* NEB 10x buffer (4 µl)
+* 1 µl restriction enzyme NgoMIV
+* 1 µ restriction enzyme AatII
+* 30 µl PCR product (geneIII with rcf 25 and AatII restriction sites)
+* 4 µl H²O
+Digestion of vector pARW089 with NarI (EheI isoschizomere) and AatII
+* 20 µl sample
+* NEB 10x buffer (2 µl)
+* 1 µl restriction enzyme NarI
+* 1 µ restriction enzyme AatII
+* 12 µl vector DNA
+* 4 µl H²O
+* 1 h at 37°C
+<b>Further Tasks:</b><br>
+* gel electrophoresis and purification of the three digested fragments
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Gel extraction from agarose gel of digested fragments mdnA, geneIII and pARW089 (strategy 2)</h3>
+<b>Investigators:</b> Stefan <br>
+<b>Aim:</b> Purification of mdnA, geneIII and pARW089 (strategy 2) to obtain an mdnA-geneIII fusion gene which can be cloned into pARW089 <br>
+<b>Time:</b> 2011-07-06,17:00-19:00<br>
+<b>Materials/Methods:</b><br>
+* PCR Clean-up Kit NucleoSpin ExtractII (Machery-Nagel) <br>
+<b>Method:</b><br>
+. Cut out visible DNA bands of the digestion product of a 0.8% and 1% agarose gel, respectivly<br>
+. Use protocol of PCR clean-up<br>
+. Two bands were cut out for the vector and genIII. Just use the lower two bands for first ligation.<br>
+<b>Results:</b><br>
+* DNA concentration geneIII: 5,4 ng/µl
+* DNA concentration mdnA: 8,2 ng/µl
+* DNA concentration pARW089: 3,8 ng/µl
+* measured by NanoDrop
+<b>Further Tasks:</b><br>
+*Repeat digestion of pARW089 (because of low concentration)and ligation of the three fragments
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">31th Labday 2011-07-07</h2>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">repeated digestion of pARW089 with NarI and AatII (strategy 2)</h3>
+<b>Investigators:</b> Sabine, Sandrina <br>
+<b>Aim:</b> Digestion of pARW089 (strategy 2) so that an mdnA-geneIII fusion gene can be cloned into pARW089 <br>
+<b>Time:</b> 2011-07-07,10:00-12:00<br>
+<b>Materials/Methods:</b><br>
+Digestion of vector pARW089 with NarI (EheI isoschizomere) and AatII
+* 20 µl sample
+* NEB 10x buffer (2 µl)
+* 1 µl restriction enzyme NarI
+* 1 µ restriction enzyme AatII
+* 3 µl vector DNA (ca. 800 ng/ µl)
+* 13 µl H²O
+* 2 1/2 h at 37°C
+* afterwards: inactivation of restriction enzymes: 20 min, 65°C
+<b>Further tasks:</b><br>
+gel electrophoresis of digested vector
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Gel electrophoresis of digested pARW089 vector(strategy 2)</h3>
+<b>Investigators:</b> Sandrina, Sabine <br>
+<b>Aim:</b>Purification of pARW089 (strategy 2) so that an mdnA-geneIII fusion gene can be cloned into pARW089 <br>
+<b>Time:</b> 2011-07-07,14:30-15:30<br>
+<b>Materials/Methods:</b><br>
+* Agarose broad range (Roth)
+* 1x TAE buffer
+* Gel Red
+* digested vector pARW089
+* DNA Ladder Gene Ruler 1kb plus(Fermentas)
+* 6x Loading Dye (Fermentas) <br>
+<b>Method:</b><br>
+<b>1. Production of a 0,5 % agarose gel</b> <br>
+* m<small>Agarose</small> = 0.25 g in 50 ml 1x TAE
+* adding 2 µl gel red <br>
+<b>2. Loading samples</b>
+* digestion product and 6x loading dye
+* pARW089 digestion product (first try, conc: 3,8 ng/µl) to verify measured concentrations<br>
+<b>3. Run</b>
+* 100 V
+* time: 00:45 h <br>
+<b>Results:</b><br>
+{| border="1" class="wikitable"
+! lane !! Sample !! Volume in µl !! Expected size in bp
+|-
+| 1 || marker || ||
+|-
+| 2 || purificated, digested vetor try 1 || 30 || ca. 10 000
+|-
+| 3 || digested pARW089 vector || 30 || ca. 10.000
+|-
+|}
+[[File: UP_versuch2_pARW_Verdau.7.7.png]] <br>
+one band was cutted out of the gel for purifacation
+<br>
+<b>Further tasks:</b><br>
+ligation of mdnA, geneIII and pARW089
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">ligation of mdnA and geneIII into pARW089(strategy 2)</h3>
+<b>Investigators:</b> Sabine<br>
+<b>Aim:</b> ligation of mdnA, geneIII, and pARW089 to get an mdnA-geneIII fusion gene in pARW089for phage display<br>
+<b>Time:</b> 2011-07-07,15:30-17:30<br>
+<b>Materials:</b><br>
+Ligation: 2 µl vector (pARW089 digested with NarI and AatII), 3 µl insert (mdnA digested with NarI and AgeI, and geneIII digested with AatII and NgoMIV), 1 µl T4 ligase, 1 µl T4 ligase NEB-buffer,over night, 16°C<br>
+<b>Further tasks:</b><br> transformation of ''E. coli'' cells(XL1-blue)
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">32th Labday 2011-07-12</h2>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Transformation of ligation mdnA, geneIII, and pARW089 (2011-07-07) for phage display (strategy 2)</h3>
+<b>Investigators:</b> Sandrina, Sabine<br>
+<b>Aim:</b>amplification of previous ligation<br>
+<b>Time:</b> 9:00-11:30<br>
+<b>Method:</b><br>
+* ligation-samples from 2011-07-07;<br>
+(ligation of mdnA, geneIII and pARW089)<br>
+protocol:
+addition of 10 µl ligation reaction to cells (XL1-blue, tet-resistance) in 1.5 ml Eppi,<br>
+incubation 25 min on ice,<br>
+heat shock 45 sec at 42°C,<br>
+incubation 2 min on ice,<br>
+addition of 750 µl LB medium,<br>
+incubation at 37 °C for 60 min in Eppendorf thermomixer at 750 rpm,<br>
+plating on LB medium with 1,5 % agar, 100 µg/ml ampicillin, 100 µg/µl tetracyclin<br>
+storage over night at 37°C<br>
+<b>Further tasks:</b><br> control cell clones
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">33th Labday 2011-07-13</h2>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">Error prone PCR (varied MnCl<small>2</small> concentration) of mdnA</h3>
+<b>Investigators:</b> Steffi, Jessica, Katharina <br>
+<b>Time:</b> 2011-06-29,14:00-16:00 <br>
+<b>Aim:</b> Random mutation of mdnA gene based on increased MnCl<small>2</small> concentration <br>
+<b>Materials and Methods:</b> see 26th Labday
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">34th Labday 2011-07-14</h2>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">Agarose gel electrophoresis of error prone PCR of mdnA from 2011-07-13 and gel extraction </h3>
+<b>Investigators:</b> Katharina <br>
+<b>Time:</b> 2011-06-29,8:00-11:00 <br>
+<b>Materials:</b>
+* Agarose broad range (Roth)
+* 1x TAE buffer Gel Red
+* 10 PCR products of mutated mdnA
+* DNA Ladder GeneRuler, 100bp plus (1:10) (Fermentas)
+* 6x Loading Dye (Fermentas)
+<b>Method:</b>
+. Production of two 1 % agarose gel
+For each gel: mAgarose = 0.5 g in 50 ml 1x TAE adding 2 µl gel red
+. Loading samples
+µl PCR product and 6x loading dye
+µl DNA Ladder GeneRuler (1:10) (Fermentas)
+. Run
+* 110 V time: 1 h
+<b>Results:</b><br>
+{| border="1" class="wikitable"
+! !! colspan="3" | gel 1 !! !! colspan="3" | gel 2
+|-
+| '''lane''' || '''Sample''' || '''Volume in µl''' || '''Expected size in bp''' || || '''Sample''' || '''Volume in µl''' || '''Expected size in bp'''
+|-
+| 1 || marker || 6 || || || marker || 6 ||
+|-
+| 2 || PCR sample 1a || 50 || 276 || || PCR sample 1b || 50 || 276
+|-
+| 3 || PCR sample 2a || 50 || 276 || || PCR sample 2b || 50 || 276
+|-
+| 4 || PCR sample 3a || 50 || 276 || || PCR sample 3b || 50 || 276
+|-
+| 5 || PCR sample 4a || 50 || 276 || || PCR sample 4b || 36 || 276
+|-
+| 6 || PCR sample 5a || 50 || 276 || || PCR sample 5b || 50 || 276
+|-
+|}
+[[File: UP_EPPCR_mdnA_2011-07-13_a.jpg |300px]] [[File: UP_EPPCR_mdnA_2011-07-30_b.jpg |300px]]
+The bands were excised and purified using the NucleoSpin Extract II (Macherey-Nagel) extraction Kit.
+<b>Further Tasks:</b><br>
+* digestion of the purified samples
+* ligation
+<br>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">Digestion of error prone PCR of mdnA from 2011-07-13 and pARW089 and agarose gel electrophoresis </h3>
+<b>Investigators:</b> Jessica <br>
+<b>Time:</b> 2011-06-29,11:00-15:00 <br>
+<b>Materials:</b>
+* Restriction enzymes: AatII, NarI
+* NEB Buffer 4
+* PCR products of random mutated mdnA(1a-5a), purified from gel
+* vector pARW089
+* Agarose broad range (Roth)
+* 1x TAE buffer
+* Gel Red
+* GeneRuler DNA Ladder Mix (1:10) (Fermentas)
+* GeneRuler 1kb DNA Ladder (Fermentas)
+* 6x Loading Dye (Fermentas)
+<b>Method:</b><br>
+<b>1. Digestion:</b>
+{| border="1" class="wikitable"
+| components || align="right" |volume of PCR products /µl|| align="right" |volume of pARW089 /µl
+|-
+| DNA || align="right" |30 || align="right" | 10
+|-
+| NEB Buffer 4(10x)|| align="right" | 4|| align="right" | 4
+|-
+|Enzyme AatII|| align="right" | 0.5 || align="right" | 0.8
+|-
+|Enzyme NarI|| align="right" | 2 || align="right" | 2
+|-
+|H<sub>2</sub>O|| align="right" | 3.5 || align="right" | 23.2
+|-
+|'''Total volume'''|| align="right" | 40 || align="right" | 40
+|}
+* digestion at 37°C for 1 h
+'''2. Production of agarose gel '''
+* 1.7% gel for PCR products: m<sub>Agarose</sub> = 0.85 g in 50 ml 1x TAE adding 2 µl gel red
+* 0.8% gel for vector: m<sub>Agarose</sub> = 0.4 g in 50 ml 1x TAE adding 2 µl gel red
+'''3. Loading samples '''
+* 40 µl digest and 8 µl 6x loading dye
+'''4. Run '''
+* 80 V, time: >1 h
+<b>Results:</b>
+''Gel 1''
+{| border="1" class="wikitable"
+! lane !! Sample !! Volume in µl !! Expected size in bp
+|-
+| M || GeneRuler DNA Ladder Mix (1:10)|| 6 ||
+|-
+| 1 || digested mdnA 1a || 48 ||
+|-
+| 2 || digested mdnA 2a || 48 ||
+|-
+| 3 || digested mdnA 3a || 48 ||
+|-
+| 4 || digested mdnA 4a || 48 ||
+|-
+| 5 || digested mdnA 5a || 48 ||
+|}
+''Gel 2''<br>
+{| border="1" class="wikitable"
+! lane !! Sample !! Volume in µl !! Expected size in bp
+|-
+| M || GeneRuler 1kb DNA Ladder || 2 ||
+|-
+| 1 || digested pARW089 || 48 ||
+|}
+[[File: UP_AG_EPPCR_digest1a-5a_2011-07-14_JE.jpg|300px]]
+[[File: UP_AG_digest_pARW089_JE_2011-07-14_JE.jpg|125px]]<br>
+* Gel excision and storage at -20°C (DNA box, orange)
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Overnight culture of PDV089</h3>
+<b>Investigators:</b> Sandrina, Sabine <br>
+<b>Aim:</b><br> control plasmid ligation (pARW089, mdnA, geneIII --> PDV089, strategy 2)
+<b>Time:</b> 2011-07-13,12:00-13:00 <br>
+<b>Materials/Methods:</b>
+* LB-medium with tet and amp
+* cell clones from over night plate
+* incubate over night at 37°C and 750 rpm
+<b>Further tasks:</b><br>
+* plasmid preparation and analytic digestion
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Repeated PCR of mdnA for phage display (strategy 1) repeated with new ordered reversed primer (with myc-tag) and geneIII</h3>
+<b>Investigators:</b> Sandrina, Sabine <br>
+<b>Time:</b> 2011-06-30,13:00-16:00 <br>
+<b>Aim:</b>
+* amplification of mdnA with SfiI restriction sites and myc (vector pARW089) to clone it into pAk100 bla KDIR <br>
+* amplificate GeneIII of pak100 with primers pf_iGEM_GenIII_NgoMIV and pr_GenIII_iGEM_AatII (phage display strategy 2), because there were two bands after digestion with NgoMIV and AatII after gel electrophoresis<br>
+<b>Materials/Methods:</b><br>
+see 2011-06-10
+changes:
+* program: 123, Thermo Hybrid, PX2
+<b>further tasks:</b><br>
+gel electrophoresis with PCR product
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">35th Labday 2011-07-15</h2>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">Ligation of mutated mdnA into pARW089</h3>
+<b>Investigators:</b> Katharina<br>
+<b>Aim:</b> create pARW089 vector with inserted mutated mdnA<br>
+<b>Time:</b> 10:00-14:00<br>
+<b>Materials:</b><br>
+Ligation: 2 µl vector (pARW089 digested with NarI and AatII), 3 µl insert (mdnA digested with NarI and AatII), 1 µl T4 ligase, 1 µl T4 ligase NEB-buffer, 4 h at room temperature<br>
+<b>Further tasks:</b><br> transformation of ''E. coli'' cells(XL1-blue)
+<br>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">Transformation of competent ''E. coli'' cells with mutated mdnA in pARW089</h3>
+<b>Investigators:</b> Katharina<br>
+<b>Aim:</b>Transformation of ligations<br>
+<b>Time:</b> 14:00-15:00<br>
+<b>Method:</b><br>
+protocol:
+addition of 2 µl ligation reaction to cells (XL1-blue) in 1.5 ml Eppi,<br>
+incubation 25 min on ice,<br>
+heat shock 45 sec at 42°C,<br>
+incubation 5 min on ice,<br>
+addition of 750 µl LB medium,<br>
+incubation at 37 °C for 60 min in Eppendorf thermomixer at 750 rpm,<br>
+plating on LB medium with 1,5 % agar, 100 µg/ml kanamycin<br>
+storage over night at 37°C<br>
+<b>Further tasks:</b><br> control cell clones
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">ligation of mdnA into pak100(strategy 1)</h3>
+<b>Investigators:</b> Sandrina, Sabine<br>
+<b>Aim:</b> ligation of mdnA into pak100 for phage display<br>
+<b>Time:</b> 2011-07-15,11:00-13:00<br>
+<b>Materials:</b><br>
+Ligation: 2 µl vector (pak100 bla KDIR, digested with SfiI), 2 µl insert (mdnA, digested with SfiI), 1 µl T4 ligase, 5 µl 2x 10x T4 ligase buffer, 20 min, room temperature<br>
+<b>Further tasks:</b><br> transformation into competent cells <br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Transformation of ligation mdnA into PAK100 bla KDIR for phage display (strategy 1)</h3>
+<b>Investigators:</b> Sandrina, Sabine<br>
+<b>Aim:</b>amplification of previous ligation<br>
+<b>Time:</b> 13.00-15:00<br>
+<b>Method:</b><br>
+* ligation-samples <br>
+(ligation of mdnA into PAK100 bla KDIR)<br>
+protocol:
+addition of 10 µl ligation reaction to cells (XL1-blue, tet-resistance) in 1.5 ml Eppi,<br>
+incubation 25 min on ice,<br>
+heat shock 45 sec at 42°C,<br>
+incubation 2 min on ice,<br>
+addition of 750 µl LB medium,<br>
+incubation at 37 °C for 60 min in Eppendorf thermomixer at 750 rpm,<br>
+plating on LB medium with 1,5 % agar, 100 µg/ml ampicillin, 100 µg/µl tetracyclin<br>
+storage over night at 37°C<br>
+<b>Further tasks:</b><br> control cell clones
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">ligation of mdnA and geneIII for strategy 2</h3>
+<b>Investigators:</b> Sabine, Sandrina<br>
+<b>Aim:</b>ligation of mdnA and geneIII to get an mdnA-geneIII fusion gene phage display<br>
+<b>Time:</b> 2011-07-15,15:00-16:00<br>
+<b>Materials:</b><br>
+Ligation: 3 µl insert mdnA digested with NarI and AgeI and geneIII digested with AatII and NgoMIV), 1 µl T4 ligase, 1 µl T4 ligase NEB-buffer, 2 µl H2O , 20 min, room temperature<br>
+<b>Results:</b><br>
+{| border="1" class="wikitable"
+! lane !! Sample !! Volume in µl !! Expected size in bp
+|-
+| M || marker, DNA ladder mix ladder mix || 10 ||
+|-
+| 1 || mdnA ligated with geneIII || || ca. 600
+|-
+|}
+[[File:Ligation mdnA-geneIII.JPG |500 px]] <br>
+<b>Further tasks:</b><br> ligation with pARW089
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">ligation of the fusion gene mdnA-geneIII into pARW089(strategy 2)</h3>
+<b>Investigators:</b> Sabine, Sandrina<br>
+<b>Aim:</b>ligation of mdnA-geneIII in pARW089 to get an mdnA-geneIII fusion gene in pARW089for phage display<br>
+<b>Time:</b> 2011-07-07,17:00-19:00<br>
+<b>Materials:</b><br>
+* Ligation: 2 µl vector (pARW089 digested with NarI and AatII), 3 µl insert, 1 µl T4 ligase, 1 µl T4 ligase NEB-buffer,over night, 16°C<br>
+* two bands were observed after mdnA-geneIII ligation (red boxes)--> ligation with pARW089 was tried with both bands
+<b>Further tasks:</b><br> transformation of ''E. coli'' cells(XL1-blue)
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">36th Labday 2011-07-16</h2>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Transformation of ligated pARW089 with mdnA-geneIII </h3>
+<b>Investigators:</b> Stefan<br>
+<b>Aim:</b>Transformation of phage display ligations (pARW089 with mdnA-geneIII) using heat shock<br>
+<b>Time:</b> 12:00-20:00<br>
+<b>Method/Materials:</b><br>
+) Take chemically competent ''E. coli'' XL1 blu cells from –80°C freezer.<br>
+) Turn on heat block to 42°C.<br>
+) use 60 µL of competent cells for each approach
+) Keep tubes on ice.<br>
+) Add 2 µL DNA solution into the ''E. coli'' cell suspension, mix by flicking the tube. Incubate on ice for 15-30 min.<br>
+) Put tubes into heat block at 42°C for 45 seconds.<br>
+) Put tubes back on ice for 2 minutes to reduce damage to the ''E. coli'' cells.<br>
+) Add 750 µL of LB with no antibiotic. Incubate tubes for 1 hour at 37°C and 750 rpm. <br>
+) spin down the cell suspension for 3 min at 6000 rcf at 4 °C , discard most of the supernatant, resuspend the bacterial pellet by pipetting and use this for spreading.<br>
+) Spread 100 ul of the resulting culture on LB agar plates with Tet/CM.<br>
+) Grow overnight at 37 °C.<br>
+) Pick colonies about 12-16 hours later.<br>
+<b>Further tasks:</b><br> picking clones and plasmid preparation
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Overnight culture of XL1 blue with mdnA in PAK100blaKDIR </h3>
+<b>Investigators:</b> Stefan<br>
+<b>Aim:</b> Picking clone 1 from plate and do an overnight culture<br>
+<b>Time:</b> 12:00-20:00<br>
+<b>Method/Materials:</b>
+<br>
+*Clone ID_1: Strain:XL1 blue, Plasmid:mdnA in PAR100bla KDIR, Clone number:1<br>
+mL of LB media were supplied with 15 µL of Tet and CM and cells were incubated at 37 °C overnight.
+<br>
+<b>Further tasks:</b><br> plasmid preparation
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">37th Labday 2011-07-17</h2>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Miniprep of ''E. coli'' overnight culture containing PAK100blaKDIR </h3>
+<b>Investigators:</b> Stefan<br>
+<b>Aim:</b> isolate the plasmid<br>
+<b>Time:</b> 12:00-20:00<br>
+<b>Method/Materials:</b>
+<br> protocol 5.1 of the NucleoSpin Plasmid Kit was used<br>
+<b>Further tasks:</b><br> sequence the plasmid<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">''E. coli'' XL1 blue stock culture containing PAK100blaKDIR with mdnA (Phage Display-strategy 1)</h3>
+<b>Investigators:</b> Stefan<br>
+<b>Aim:</b> set up a stock culture<br>
+<b>Time:</b> 12:00-20:00<br>
+<b>Method/Materials:</b>
+<br>2 x 700 µL culture were mixed with 300 µL of glycerol and stored in -80 °C freezer<br>
+<b>Further tasks:</b><br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">overnight culture of ''E. coli'' Xl blue m. Ligation089jessi, u.Ligationvector089jessi,u.Ligationu.BandemdnAgeneIIIpARW089, mLigatiojnm.BandemdnAgeneIIIpARW089</h3>
+<b>Investigators:</b> Stefan<br>
+<b>Aim:</b> pick two clones each and inoculate them overnight in 15 mL LB supplied with 15 µL Tet and Amp<br>
+<b>Time:</b> 12:00-20:00<br>
+<b>Method/Materials:</b>
+<br>
+<b>Further tasks:</b><br>plasmid preparation <br>
+<h2 style="background-color: rgb(240, 20, 70);">38th Labday 2011-07-18</h2>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">overnight culture of ''E. coli'' XL-1 blue containing mutated mndA in parW089</h3>
+<b>Investigators:</b> Katharina<br>
+<b>Aim:</b> Picking 5 clones from each plate and do an overnight culture<br>
+<b>Time:</b> 14:00-15:00<br>
+<b>Method/Materials:</b>
+<br> ''E. coli'' Strain:XL1 blue, Plasmid:mdnA in parW089<br>
+mL of LB media were supplied with 5 µL of Kan and cells were incubated at 37 °C overnight.
+<br>
+<b>Further tasks:</b><br> plasmid preparation
+<br>
+<h3 style="background-color: rgb(100, 150, 100); font-weight: bold;">Dual-Tranformation of XL1-blu (''E. coli'') with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla</h3>
+<b>Investigators:</b> Sascha <br>
+<b>Aim:</b><br>
+* Dual-Transformation of XL1-blue to test the in-vivo selection system (pTEV-SCS1 + pJC354_ssTorA_NheI_CS-TEV_XhoI_bla) <br>
+<b>Materials:</b><br>
+* Plasmids:<br>
+** pTEV-SCS1 (TEV protease, res. against kanamycin)
+** pJC354_ssTorA_NheI_CS-TEV_XhoI_bla (res. against chloramphenicol) <br>
+* competent cells XL1-blue
+* LB media and diffenet antibiotics
+<b> Used method: </b>
+*standard protocol for heat shock transformation
+'''Output:''' transformed cultures with plasmids
+* XL1-blue without plasmid (negativ control - no growth over night)
+* XL1-blue transformed with pTEV-SCS1 - growth with kanamycin in LB media
+* XL1-blue transformed with pJC354_ssTorA_NheI_CS-TEV_XhoI_bla - growth with chloramphenicol in LB media
+* XL1-blue transformed with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla - growth with kanamycin and chloramphenicol in LB media
+<b>Further tasks:</b><br>
+* reinfect media with cultures for new growth period and distribution to LB Agar with different antibiotic concentrations
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Test digestion of ligations for strategies 1 and 2 </h3>
+<b>Investigators:</b> Sandrina <br>
+<b>Aim:</b>control if liagation of geneIII and mdnA into pARW089 (strategy 2) and ligation of mdnA into PAK100 bla KDIR worked<br>
+<b>Time:</b> 15:30-19:00<br>
+<b>Materials/Methods:</b><br>
+Strategy 1:
+* 0,5 µl SfiI
+* 2 µl 10x buffer 4 (NEB)
+* 0,2 µl BSA
+* 10 µl vector DNA
+* 13,3 µl H2O
+incubate for 1 h at 50°C
+Strategy 2:
+* 0,5 µl XbaI
+* 0,5 µl SpeI
+* 2 µl 10x buffer 4 (NEB)
+* 0,2 µl BSA
+* 10 µl vector DNA
+* 12,8 µl H2O
+incubate for 1 h at 37°C
+<br>
+<b>Results:</b><br>
+*expected bands: strategy 1: ca. 5000 bp and 200bp
+*expected bands: strategy 2: ca. 5000 bp, 4000 bp, 600 bp and 200
+observed bands:
+*srtategy 1: ca. 5000 and 800 bp
+*strategy 2: ca. 5000 and 200 bp
+<b>Further tasks:</b><br>
+* repeat ligations
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">39th Labday 2011-07-19</h2>
+<h3 style="background-color: rgb(100, 150, 100); font-weight: bold;">Resistance test of the Dual-Transformation of XL1-blue (''E. coli'') with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla</h3>
+<b>Investigators:</b> Stefan and Sebastian <br>
+<b>Aim:</b><br>
+* Dual-Transformation of XL1-blue to test the in-vivo selection system (pTEV-SCS1 + pJC354_ssTorA_NheI_CS-TEV_XhoI_bla) <br>
+<b>Materials:</b><br>
+*Overnight cultures
+** XL1-blue transformed with pTEV-SCS1 - growth with kanamycin in LB media
+** XL1-blue transformed with pJC354_ssTorA_NheI_CS-TEV_XhoI_bla - growth with chloramphenicol in LB media
+** XL1-blue transformed with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla - growth with kanamycin and chloramphenicol in LB media
+* LB media and different antibiotics
+<b> Used method: </b>
+* growth over night
+* set OD (600 nm) after measurement cells were dilutes to OD 0,1, transformed XL1-blue grow again to OD 0,5. Out of this approach a dilution to OD= 0.002 was done and platted to LB agar with kanamycin, IPTG and different concentrations of ampicillin (range form 0-800 ng/ml ampicillin)
+'''Output:'''
+* survival of dual transformed cells...
+[[File: UP_resistance test_agar plattes with TEV and TorAbla_11-7-20_STW_picture1.jpg|300px|]]
+[[File: UP_resistance test_agar plattes with TEV _11-7-20_STW_picture1.jpg|300px|]]<br>
+<b>Further tasks:</b><br>
+There was no growth on the TorAbla plates due to the choice of the wrong antibiotic. This was set up again as well as well as the double transformed cells.
+<br>
+<h3 style="background-color: rgb(100, 150, 100); font-weight: bold;">Stock cultures of XL1-blue(''E. coli'')with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla and of XL1-blue(''E. coli'')with pTEV-SCS1 and of XL1-blue(''E. coli'')pJC354_ssTorA_NheI_CS-TEV_XhoI_bla </h3>
+<b>Investigators:</b> Stefan <br>
+<b>Aim:</b><br>
+* make stock cultures <br>
+<b>Materials:</b><br>
+*Overnight cultures were used. 700 µL culture were spun down to remove antibiotics. The pellets is resuspended in 700 µL LB media and 300 µL glycerol.
+<b> Used method: </b>
+<b>Further tasks:</b><br>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">Design of Primers for BioBrick mdnA and mdn-Cluster </h3>
+<b>Investigators:</b> Nicole, Nadine <br>
+<b>Aim:</b> Design primers for mdnA and mdn-Cluster(from pARW089) for BioBricks pBAD_mdn_Cluster, pSB_mdn_Cluster and pSB_mdnA <br>
+<b>Time:</b> 2011-07-19,14:00-17:00<br>
+<b>Materials:</b><br>
+* Geneious Pro 5.1.7<br>
+<b>Method:</b><br>
+* Primer design
+** melting temperature based on 4+2 method (for mdnA-overlapping part)
+** adding sequences for restriction sites (considering reading frame)
+** Check self-complementarity and melting temperature using Oligo Calc
+<b>Plan:</b><br>
+* mdn-Cluster BioBricks
+[[File: UP_BioBrick_plan_mdn_cluster_2011_07_19.png|600px]]
+* mdnA BioBrick
+[[File: UP_BioBrick_plan_mdnA_2011_07_19.png|600px]]
+<br>
+<b>Results:</b><br>
+Primer sequences <br>
+[[File: UP_Primer_design_mdn_cluster_mdnA_2011-07-19.png|600px]]
+<br>
+<b>Further tasks:</b><br>
+* do PCR of mdnA
+<br>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">Miniprep of ''E. coli'' Xl blue containing mutated mndA in parW089 </h3>
+<b>Investigators:</b> Niels
+<br>
+<b>Aim:</b> 25x overnight cultures :
+- miniprep
+<b>Time:</b> 2011-07-19,12:00-16:00<br>
+<b>Materials:</b><br>
+* ''E. coli'' Xl blue <br>
+<b>Kit:</b><br>
+* Nucleic Acid and Protein Purification
+** Support protocol for NucleoSpin Plasmid
+<b>Results:</b><br>
+Nanodrop: <br>
+[[File: UP_Nanodrop_2011-07-19_NW.png|700px]]<br>
+<b>Further task:</b><br>
+* sequencing
+* digest <br>
+<h2 style="background-color: rgb(240, 20, 70);">40th Labday 2011-07-20</h2>
+<h3 style="background-color: rgb(100, 150, 100); font-weight: bold;">Resistance test of the Dual-Transformation of XL1-blue (''E. coli'') with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla</h3>
+<b>Investigators:</b> Stefan <br>
+<b>Aim:</b><br>
+* overnight cultures for resistance test <br>
+<b>Materials:</b><br>
+*Overnight cultures of
+** XL1-blue transformed with pJC354_ssTorA_NheI_CS-TEV_XhoI_bla - growth with chloramphenicol in LB media
+** XL1-blue transformed with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla - growth with kanamycin and chloramphenicol in LB media
+<b> Used method: </b>
+<br>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">Restriction enzyme digestion pARW089 and pARW071</h3>
+<b>Investigators:</b> Katharina, Nadine<br>
+<b>Time:</b> 2011-07-21,11:00-12:00 <br>
+<b>Aim:</b> Restriction enzyme digestion of pARW089 and pARW071<br>
+<b>Materials:</b><br>
+* Restriction enzymes: AatII, EheI (both from Elke)
+* NEB Buffer 4
+* Plasmids: pARW089, pARW071
+<b>Problem:</b>
+* AatII was empty (ordered new batch for tomorrow)
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">overnight culture of ''E. coli'' Xl blue m. ligation mdnA and geneIII into pARW089 </h3>
+<b>Investigators:</b> Sandrina<br>
+<b>Aim:</b> repeated test digestion of ligation mdnA and geneIII into pARW089 from 2011-07-12/13<br>
+<b>Time:</b> 16:30-18:00<br>
+<b>Method/Materials:</b>
+*LB medium
+<b>Further tasks:</b><br>plasmid preparation and test digestion <br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Repeated PCR of mdnA and gene III for phage display (strategy 1+2)</h3>
+<b>Investigator:</b> Sandrina <br>
+<b>Time:</b> 2011-07-26,9:00-11:00 <br>
+<b>Aim:</b>
+* amplification of mdnA with SfiI restriction sites (strategy 1)<br>
+* amplification of mdnA with NarI and AgeI and rfc 25 restriction sites (strategy 2)<br>
+* amplificate GeneIII with NgoMIV and AatII and rfc 25 restriction sites (strategy 2)<br>
+<b>Primer:</b><br>
+* primer: pf_sfi-mdnA_2 and pr_sfi_mdnA_myc (mdnA, strategy 1)<br>
+* primer: pf_mdnA_iGEM_EheI and pr_mdnA_iGEM_AatII (mdnaA, strategy 2)<br>
+* primer: pf_geneIII_NgoMIV and pr_geneIII_iGEM_AatII (geneIII, strategy 2)<br>
+<b>Reaction Components:</b><br>
+* 5 µl Vector pARW089
+* 0,25 µl OneTaq Polymerase
+* 1 µl dNTPs
+* 1 µl per primer
+* 5 µl 5x PCR Buffer
+* 37,75 µl DNase free water
+<br>
+* purification of PCR fragments with QIAquick Gel Extraction Kit (250)
+<b>Further tasks:</b><br>
+* digestion of PCR products
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">digestion of PCR products and vectors (strategy 1+2)</h3>
+<b>Investigator:</b> Sandrina<br>
+<b>Time:</b> 2011-07-26,11:00-13:00 <br>
+<b>Aim:</b>
+* digestion of mdnA and gene III for getting an mdnA-geneIII fusion gene with rfc25 restrition sites after ligation
+* digestion of pARW089 for ligation of mdnA/geneIII fusion gene into it (strategy 2)<br>
+* digestion of pak100blaKDIR and and mdnA to enable ligation
+<b>Time:</b> 2011-07-26,14:00-17:30<br> <br>
+<b>digestion enzymes:</b><br>
+* digestion of mdnA (strategy 2) with NarI and AgeI
+* digestion of geneIII (strategy 2) with NgoMIV and AatII
+* digestion of pARW089 (strategy 2) with NarI and AatII
+* digestion of pak100blaKDIR (strategy 1) with SfiI
+* digestion of mdnA (strategy 1) with SfiI
+<b>reaction components:</b>
+* 4 µl NEB 10x buffer
+* 1 µl per restriction enzyme
+* 30 µl PCR product / 5 µl vector
+* 4 µl H²O
+* BSA (only Sfi digestion)
+<b>reaction conditions:</b>
+* 1 h for PCR fragments
+* 3 h for plasmids
+* 50°C (SfiI digestion)
+* 37°C NarI, AgeI, NgoMIV and AatII digestion
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">ligation of mdnA and geneIII into digested pARW089(strategy 2)</h3>
+<b>Investigators:</b> Sandrina<br>
+<b>Aim:</b> get phage display vector pPDV089<br>
+<b>Time:</b> 2011-07-20,14:00-16:00<br>
+<b>Materials:</b><br>
+* 6 µl digested vector pARW089 (NarI, AatII)
+* 1 µl geneIII
+* 1 µl mdnA
+vector and insert should be added in ratio 1:3
+* 2 µl 10x T4 Ligase Buffer
+* 1 µl T4 Ligase
+* 9 µl water
+* incubation over night at 16°C
+<b>Further Tasks:</b> transormation of competent cells
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">ligation of mdnA into pak100blaKDIR (stategy 1)</h3>
+<b>Investigator:</b> Sandrina<br>
+<b>Aim:</b> generate phage display vector pPDV100 (strategy 1)<br>
+<b>Time:</b> 2011-07-20,14:00-16:00<br>
+<b>Method/Materials:</b>
+* 3 µl (ca 90 ng) Sfi-digested vector pak100
+* 1 µl (ca 20 ng) Sfi-digested PCR fragment mdnA
+* 2 µl 10x T4 Ligase Buffer
+* 1 µl T4 Ligase
+* 13 µl water
+* incubate over night at 16°C
+<b>Further Tasks:</b> transformation of competent cells
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">41th Labday 2011-07-21</h2>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Test digestion of ligation for strategy 2 </h3>
+<b>Investigators:</b> Sandrina <br>
+<b>Aim:</b>repeated test digestion of ligation mdnA and geneIII into pARW089 from 2011-07-12/13<br>
+<b>Time:</b> 15:30-19:00<br>
+<b>Materials/Methods:</b><br>
+Strategy 2:
+* 0,5 µl XbaI
+* 0,5 µl SpeI
+* 2 µl 10x buffer 4 (NEB)
+* 0,2 µl BSA
+* 10 µl vector DNA
+* 12,8 µl H2O
+incubate for 1 h at 37°C
+<br>
+<b>Results:</b><br>
+{| border="1" class="wikitable"
+! lane !! Sample !! Volume in µl !! Expected size in bp
+|-
+| M || marker, DNA ladder mix, fermentas || 2 ||
+|-
+| 1-20 || digested ligations || 6 || ca. 5000 bp, 4000 bp, 600 bp and 200
+|-
+|}
+[[File:Test verdau PDV089.png |400 px]] <br>
+[[File:Test verdau 2 PDV089.png |400 px]] <br>
+<b>Further tasks:</b><br>
+sequencing of clone 6
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Transformation of ligated pARW089 with mdnA-geneIII and ligated PAK100 bla KDIR with mdna</h3>
+<b>Investigators:</b> Sandrina<br>
+<b>Aim:</b>Transformation of phage display ligations (strategy 1 and2) (2011-7-20) using heat shock<br>
+<b>Time:</b> 10:00-12:00<br>
+<b>Method/Materials:</b>
+<br>1) Take chemically competent ''E. coli'' XL1 blu cells from –80°C freezer.<br>
+) Turn on heat block to 42°C.<br>
+) use 60 µL of competent cells for each approach
+) Keep tubes on ice.<br>
+) Add 2 µL DNA solution into the ''E. coli'' cell suspension, mix by flicking the tube. Incubate on ice for 15-30 min.<br>
+) Put tubes into heat block at 42°C for 45 seconds.<br>
+) Put tubes back on ice for 2 minutes to reduce damage to the ''E. coli'' cells.<br>
+) Add 750 µL of LB with no antibiotic. Incubate tubes for 1 hour at 37°C and 750 rpm. <br>
+) spin down the cell suspension for 3 min at 6000 rcf at 4 °C , discard most of the supernatant, resuspend the bacterial pellet by pipetting and use this for spreading.<br>
+) Spread 100 ul of the resulting culture on LB agar plates with Tet/CM.<br>
+) Grow overnight at 37 °C.<br>
+) Pick colonies about 12-16 hours later.<br>
+<b>Further tasks:</b><br> picking clones and plasmid preparation
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">42th Labday 2011-07-22</h2>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">Restriction enzyme digestion pARW089 and pARW071 AND trouble-shouting</h3>
+<b>Investigators:</b> Katharina, Nadine<br>
+<b>Time:</b> 2011-07-22,10:00-15:00 <br>
+<b>Aim:</b> Restriction enzyme digestion of pARW089 and pARW071<br>
+<b>Materials:</b><br>
+* Restriction enzymes: AatII, NarI
+* NEB Buffer 4
+* Plasmids: pARW089, pARW071
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">43th Labday 2011-07-23</h2>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">Restriction enzyme digestion pARW089 (different incubation times), plasmid purification and ligation with EP-PCR mdnA</h3>
+<b>Investigators:</b> Nadine<br>
+<b>Time:</b> 2011-07-22,14:30-20:30 <br>
+<b>Aim:</b> Restriction enzyme digestion of pARW089 with different incubation times to test best digestion conditions, problems with the digestion of pARW089 in the last trials (-->gel excision and ligation if it worked out <br>
+<b>Materials:</b>
+* Restriction enzymes: AatII, NarI, control: XmaI
+* NEB Buffer 4
+* PCR products of random mutated mdnA(1a-5a), purified from gel
+* vector pARW089
+* Agarose broad range (Roth)
+* 1x TAE buffer
+* Gel Red
+* GeneRuler 1kb DNA Ladder (Fermentas)
+* 6x Loading Dye (Fermentas)
+<b>Method:</b><br>
+<b>1. Digestion:</b>
+{| border="1" class="wikitable"
+| components || align="right" |volume of pARW089 /µl
+|-
+| DNA || align="right" | 5
+|-
+| NEB Buffer 4(10x)|| align="right" | 4
+|-
+|Enzyme AatII|| align="right" | 0.8
+|-
+|Enzyme NarI|| align="right" | 2
+|-
+|H<sub>2</sub>O|| align="right" | 28.2
+|-
+|'''Total volume'''|| align="right" | 40
+|}
+<b>2. Digestion: control</b>
+{| border="1" class="wikitable"
+| components || align="right" |volume of pARW089 /µl
+|-
+| DNA || align="right" | 5
+|-
+| NEB Buffer 4(10x)|| align="right" | 4
+|-
+|Enzyme XmaI|| align="right" | 1
+|-
+|BSA|| align="right" | 0.2
+|-
+|H<sub>2</sub>O|| align="right" | 29.2
+|-
+|'''Total volume'''|| align="right" | 40
+|}
+* digestion at 37°C for 1 h
+'''3. Production of agarose gel '''
+* 0.8% gel for vector: m<sub>Agarose</sub> = 0.4 g in 50 ml 1x TAE adding 4 µl gel red
+'''4. Loading samples '''
+* 40 µl digest and 8 µl 6x loading dye
+'''5. Run '''
+* 110 V, time: >40 min
+<b>Results:</b>
+''Gel ''<br>
+{| border="1" class="wikitable"
+! lane !! Sample !! Volume in µl !! Expected size in bp
+|-
+| M || GeneRuler 1kb DNA Ladder || 2 || 10296, 116
+|-
+| 1 || digested pARW089 1 hr || 48 || 10296, 116
+|-
+| 2 || digested pARW089 2 hrs || 48 || 10296, 116
+|-
+| 3 || digested pARW089 2.5 hrs || 48 || 10296, 116
+|-
+| 4 || digested pARW089 XmaI 3 hrs || 48 || 3270, 7142
+|-
+| 5 || digested pARW089 3 hrs || 48 || 10296, 116
+|-
+| 6 || digested pARW089 undigested|| 48 ||
+|-
+|}
+[[File: UP_AG_digest_pARW089_different_incubation_times_2011-07-23_JE.jpg|250px]]<br>
+''' 6. Gel excision '''
+* lane 4 and 6, band: 10296, respectively
+''' 7. Ligation '''
+Ligation: 3 µl vector (pARW089 digested with NarI and AatII, incubation 2.5 hrs and 3 hrs), 3 µl insert (mdnA digested with NarI and AatII from 2011-07-29), 1 µl T4 ligase, 1 µl T4 ligase NEB-buffer, overnight at 16°C<br>
+samples:
+* pARW089 (dig. 2.5 hrs) + mdnA 1A
+* pARW089 (dig. 2.5 hrs) + mdnA 2A
+* pARW089 (dig. 2.5 hrs) + mdnA 3A
+* pARW089 (dig. 2.5 hrs) + mdnA 4A
+* pARW089 (dig. 2.5 hrs) + mdnA 5A
+* pARW089 (dig. 3 hrs) + mdnA 1A
+* pARW089 (dig. 3 hrs) + mdnA 2A
+* pARW089 (dig. 3 hrs) + mdnA 3A
+* pARW089 (dig. 3 hrs) + mdnA 4A
+* pARW089 (dig. 3 hrs) + mdnA 5A
+<b>Further tasks:</b><br> transformation of ''E. coli'' cells(XL1-blue)
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">creation of pPDV089 in Genious</h3>
+<b>Investigators:</b> Sandrina, Sabine<br>
+<b>Aim:</b> getting a model of pPDV089 for sequence alignment with purified pPDV089 plasmids from clones<br>
+<b>Time:</b> 09.00-11.00<br>
+<b>Method/Materials:</b>
+<br>software Genious<br>
+<b>Further tasks:</b><br>
+perform alignment
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">sending pPDV089 from positive clone to GATC for sequencing</h3>
+<b>Investigators:</b> Sabine<br>
+<b>Aim:</b> get sequence of generated phage display vector pPDV089 (strategy 2) to control the ligation of digested pARW089 with digested mdnA and gene III<br>
+<b>Method/Materials:</b>
+<br>10 µl of purified pPDV089 and 20 µl primer sf_mdna_1 were sent to GATC<br>
+<b>Further tasks:</b><br>
+perform alignment
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">44th Labday 2011-07-24</h2>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">Transform ligation from 42th Labday into ''E. coli'' XL blue 1 cells</h3>
+<b>Investigators:</b> Stefan<br>
+<b>Time:</b> 2011-07-22,11:00-15:00 <br>
+<b>Aim:</b> cells containing the desired vectors<br>
+<b>Materials/Methods:</b>
+<br>1)Take chemically competent ''E. coli'' XL1 blu cells from –80°C freezer.<br>
+) Turn on heat block to 42°C. <br>
+) use 60 µL of competent cells for each approach<br>
+) Keep tubes on ice.<br>
+) Add 2 µL DNA solution into the ''E. coli'' cell suspension, mix by flicking the tube. Incubate on ice for 15-30 min. <br>
+) Put tubes into heat block at 42°C for 45 seconds. <br>
+) Put tubes back on ice for 2 minutes to reduce damage to the ''E. coli'' cells. <br>
+) Add 750 µL of LB with no antibiotic. Incubate tubes for 1 hour at 37°C and 750 rpm. <br>
+) spin down the cell suspension for 3 min at 6000 rcf at 4 °C , discard most of the supernatant, resuspend the bacterial pellet by pipetting and use this for spreading. <br>
+) Spread 100 ul of the resulting culture on LB agar plates with Tet/CM. 11) Grow overnight at 37 °C. 12) Pick colonies about 12-16 hours later.<br>
+<h2 style="background-color: rgb(240, 20, 70);">45th Labday 2011-07-25</h2>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">overnight culture of ''E. coli'' XL-1 blue containing mutated mndA in parW089</h3>
+<b>Investigators:</b> Steffi<br>
+<b>Aim:</b> Picking 1 clone from each plate (Ligation pARW089 mdnA 2hrs 1A-5A, Ligation pARW089 mdnA 3hrs 1A-5A) and do an overnight culture<br>
+<b>Time:</b> 16:00-17:00<br>
+<b>Method/Materials:</b>
+<br> ''E. coli'' Strain: XL1 blue, Plasmid:mdnA in parW089<br>
+mL of LB media were supplied with 5 µL of Kan and cells were incubated at 37 °C overnight.
+<b>Further tasks:</b><br> plasmid preparation<br>
+<h2 style="background-color: rgb(240, 20, 70);">46th Labday 2011-07-26</h2>
+<h3 style="background-color: rgb(254, 122, 122); font-weight: bold;">Miniprep of ''E. coli'' Xl blue containing mutated mndA in parW089 </h3>
+<b>Investigators:</b> Steffi
+<b>Aim:</b> 10x overnight cultures :
+- miniprep
+<b>Time:</b> 2011-07-26,09:00-16:00<br>
+<b>Materials:</b><br>
+* ''E. coli'' Xl blue <br>
+<b>Kit:</b><br>
+* Nucleic Acid and Protein Purification
+** Support protocol for NucleoSpin Plasmid
+<b>Further task:</b><br>
+* sequencing
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Alignment of sequenced pPDV089 and model of pPDV089 </h3>
+<b>Investigator:</b> Sabine <br>
+<b>Time:</b> 2011-07-26,10:30-11:30 <br>
+<b>Aim:</b> control of generated phage display vector pPDV089<br>
+<b>Materials/Methods:</b><br>
+* file sent from GATC
+* software Genious
+<b>Results:</b><br>
+* 2 mutations in geneIII
+* the last 2 aminoacids of myc are absent
+<b>Further task:</b><br>
+* repeat PCR of all fragments using polymerase S (not OneTaq as before)
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Repeated PCR of mdnA and gene III for phage display (strategy 1+2)</h3>
+<b>Investigator:</b> Sabine <br>
+<b>Time:</b> 2011-07-26,11:30-13:30 <br>
+<b>Aim:</b>
+* amplification of mdnA with SfiI restriction sites (strategy 1)<br>
+* amplification of mdnA with NarI and AgeI restriction sites (strategy 2)<br>
+* amplificate GeneIII with NgoMIV and AatII restriction sites (strategy 2)<br>
+<b>Primer:</b><br>
+* primer: pf_sfi-mdnA_2 and pr_sfi_mdnA_myc (mdnA, strategy 1)<br>
+* primer: pf_mdnA_iGEM_EheI and pr_mdnA_iGEM_AatII (mdnaA, strategy 2)<br>
+* primer: pf_geneIII_NgoMIV and pr_geneIII_iGEM_AatII (geneIII, strategy 2)<br>
+<b>Reaction Components:</b><br>
+* 5 µl Vector pARW089
+* 0,25 µl Taq Polymerase S (BioScience)
+* 1 µl dNTPs
+* 1 µl per primer
+* 5 µl 10x PCR Buffer S
+* 37,75 µl DNase free water
+<br>
+* purification of PCR fragments with QIAquick Gel Extraction Kit (250)
+<b>Further tasks:</b><br>
+* digestion
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">digestion of PCR products and vectors (strategy 1+2)</h3>
+<b>Investigator:</b> Sabine<br>
+<b>Aim:</b>
+* digestion of mdnA and gene III for getting an mdnA-geneIII fusion gene with rfc25 restrition sites after ligation
+* digestion of pARW089 for ligation of mdnA/geneIII fusion gene into it (strategy 2)<br>
+* digestion of pak100blaKDIR and and mdnA to enable ligation
+<b>Time:</b> 2011-07-26,14:00-17:30<br> <br>
+<b>digestion enzymes:</b><br>
+* digestion of mdnA (strategy 2) with NarI and AgeI
+* digestion of geneIII (strategy 2) with NgoMIV and AatII
+* digestion of pARW089 (strategy 2) with NarI and AatII
+* digestion of pak100blaKDIR (strategy 1) with SfiI
+* digestion of mdnA (strategy 1) with SfiI
+<b>reaction components:</b>
+* 4 µl NEB 10x buffer
+* 1 µl per restriction enzyme
+* 30 µl PCR product / 5 µl vector
+* 4 µl H²O
+* BSA (only Sfi digestion)
+<b>reaction conditions:</b>
+* 1 h for PCR fragments
+* 3 h for plasmids
+* 50°C (SfiI digestion)
+* 37°C NarI, AgeI, NgoMIV and AatII digestion
+<b>Further Tasks:</b><br>
+* gel electrophoresis for purification of the digested fragments and vectors
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">gel electrophoresis of digested fragments and vectors</h3>
+<b>Investigator:</b> Sabine<br>
+<b>Aim:</b> control PCR and digestion
+<b>Time:</b> 2011-07-06,18:00-19:00<br>
+<b>Results:</b>
+sizes of bands
+* pak100blaKDIR (SfiI, strategy 1): ca 4,5 kp
+* pARW089 (NarI and AatII, strategy 2): ca 10,5 kb
+* mdnA (SfiI, strategy 1): ca 250 bp
+* mdnA (NarI and AgeI, stategy 2): ca 200 bp
+* geneIII (NgoMIV and AatII): no band
+<br>
+* purification with QIAquick Gel Extraction Kit (250)
+<b>Further Tasks:</b><br>
+* repeat PCR of geneIII
+* ligation
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Repeated PCR gene III for phage display (strategy 2)</h3>
+<b>Investigator:</b> Sabine <br>
+<b>Time:</b> 2011-07-26,19:00-19:30 <br>
+<b>Aim:</b>
+* amplificate GeneIII with NgoMIV and AatII restriction sites (strategy 2)<br>
+<b>Primer:</b><br>
+* primer: pf_geneIII_NgoMIV and pr_geneIII_iGEM_AatII (geneIII, strategy 2)<br>
+<b>Reaction Components:</b><br>
+* 5 µl pak100blaKDIR
+* 0,25 µl Taq Polymerase S (BioScience)
+* 1 µl dNTPs
+* 1 µl per primer
+* 5 µl 10x PCR Buffer S
+* 37,75 µl DNase free water
+<b>Further tasks:</b><br>
+* purification
+* digestion
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">47th Labday 2011-07-27</h2>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">digestion of PCR product geneIII (strategy 2)</h3>
+<b>Investigator:</b> Sabine<br>
+<b>Aim:</b> digestion of gene III for getting an mdnA-geneIII fusion gene with rfc25 restrition sites after ligation
+<b>Time:</b> 2011-07-26,09:30-10:30<br>
+<b>reaction components:</b>
+* 4 µl NEB 10x buffer
+* 1 µl per restriction enzyme (NgoMIV and AatII
+* 30 µl PCR product
+* 4 µl H²O
+<b>reaction conditions:</b>
+* 1 h, 37°C
+<b>Further Tasks:</b><br>
+* gel electrophoresis for purification of the digested geneIII
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">gel electrophoresis of digested geneIII</h3>
+<b>Investigator:</b> Sabine<br>
+<b>Aim:</b> control PCR and digestion<br>
+<b>Time:</b> 2011-07-27,10:30-11:30<br>
+<b>Method/Materials:</b>
+* 1% agarose gel
+* GeneRuler DNA Ladder Mix (SM0331)
+* 5x loading dye<br>
+* purification with QIAquick Gel Extraction Kit (250)
+<b>Results:</b> size of band: ca 550 bp<br>
+<b>Further Tasks:</b> ligation
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">ligation of mdnA into pak100blaKDIR (stategy 1)</h3>
+<b>Investigator:</b> Sabine<br>
+<b>Aim:</b> generate phage display vector pPDV100 (strategy 1)<br>
+<b>Time:</b> 2011-07-27,11:30-12:30<br>
+<b>Method/Materials:</b>
+* 3 µl (ca 90 ng) Sfi-digested vector pak100
+* 1 µl (ca 20 ng) Sfi-digested PCR fragment mdnA
+* 2 µl 10x T4 Ligase Buffer
+* 1 µl T4 Ligase
+* 13 µl water
+* 1 h, room temperature
+<b>Further Tasks:</b> transormation
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Transformation of generated pPDV100 (strategy 1)</h3>
+<b>Investigators:</b> Sabine<br>
+<b>Aim:</b>amplification of pPDV100<br>
+<b>Time:</b> 12:30-14:00<br>
+<b>Method:</b><br>
+* addition of 10 µl ligation reaction to XL1-blue cells<br>
+* incubation 25 min on ice,<br>
+* heat shock 45 sec at 42°C,<br>
+* incubation 2 min on ice,<br>
+* addition of 750 µl LB medium,<br>
+* incubation 60 min at 37 °C and 750 rpm<br>
+* plating on agar plates containing 100 µg/ml chloramphenicol<br>
+* storage over night at 37°C<br>
+<b>Further tasks:</b><br> control cell clones
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">48th Labday 2011-07-28</h2>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">overnight culture of 10 picked clones of XL blue cells transformed with pPDV100</h3>
+<b>Investigators:</b> Sabine<br>
+<b>Aim:</b> amplification and purification of generated phage display vector pPDV100 for test digestion and sequencing<br>
+<b>Time:</b> 10:00-10:30<br>
+<b>Method/Materials:</b>
+ml LB medium per clone containining 100 µg/ml chloramphenicol
+storage over night at 37°C and 800 rpm<br>
+<b>Further tasks:</b>
+plasmid preparation, test digestion and sequencing<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">ligation of mdnA and geneIII (strategy 2)</h3>
+<b>Investigators:</b> Sabine<br>
+<b>Aim:</b> get mdnA/geneIII fusion gene for cloning into pARW089<br>
+<b>Time:</b> 2011-07-28,11:00-12:30<br>
+<b>Materials:</b><br>
+* 5 µl (ca 25 ng) digested geneIII (NgoMIV, AatII)
+* 3 µl (ca 30 ng) digested mdnA (NarI, AgeI)
+* 2 µl 10x T4 Ligase Buffer
+* 1 µl T4 Ligase
+* 9 µl water
+* 1 h, room temperature
+<b>Further Tasks:</b> ligation of mdnA/geneIII into digested pARW089
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">ligation of mdnA/geneIII into digested pARW089(strategy 2)</h3>
+<b>Investigators:</b> Sabine<br>
+<b>Aim:</b> get phage display vector pPDV089<br>
+<b>Time:</b> 2011-07-28,12:30-13:00<br>
+<b>Materials:</b><br>
+* 6 µl (ca 70 ng) digested vector pARW089 (NarI, AatII)
+* 1 µl (ca 5 ng) fusion gene mdnA/geneIII
+* 2 µl 10x T4 Ligase Buffer
+* 1 µl T4 Ligase
+* 10 µl water
+* 1 h, room temperature
+<b>Further Tasks:</b> transormation
+<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Transformation of generated pPDV089 (strategy 2)</h3>
+<b>Investigators:</b> Sabine<br>
+<b>Aim:</b> amplification of pPDV089<br>
+<b>Time:</b> 13:30-15:00<br>
+<b>Method:</b><br>
+* addition of 10 µl ligation reaction to XL1-blue cells<br>
+* incubation 25 min on ice,<br>
+* heat shock 45 sec at 42°C,<br>
+* incubation 2 min on ice,<br>
+* addition of 750 µl LB medium,<br>
+* incubation 60 min at 37 °C and 750 rpm<br>
+* plating on agar plates containing 100 µg/ml tetracyclin and 100 µg/µl ampicillin<br>
+* storage over night at 37°C<br>
+<b>Further tasks:</b><br> control cell clones
+<br>
+<h2 style="background-color: rgb(240, 20, 70);">49th Labday 2011-07-29</h2>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">Miniprep of ''E. coli'' overnight culture containing pPDV100 </h3>
+<b>Investigators:</b> Sabine<br>
+<b>Aim:</b> purification of pPDV100 for test digestion and sequencing<br>
+<b>Time:</b> 10:00-11:00<br>
+<b>Method/Materials:</b> see protocol 5.1 of the NucleoSpin Plasmid Kit<br>
+<b>Further tasks:</b> test digestion<br>
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">gel electrophoresis of digested pPDV100</h3>
+<b>Investigator:</b> Sabine<br>
+<b>Aim:</b> control ligated pPDV100<br>
+<b>Time:</b> 2011-07-29,11:30-12:30<br>
+<b>Method/Materials:</b>
+% agarose gel, GeneRuler DNA Ladder Mix (SM0331), 5x loading dye<br>
+<b>Results:</b> ligation of mdnA into pak100 has not worked<br>
+<b>Further Tasks:</b> control all working steps, repeat cloning
+<h3 style="background-color: rgb(238, 221, 130); font-weight: bold;">creation of pPDV100 in Genious</h3>
+<b>Investigators:</b> Sandrina, Sabine<br>
+<b>Aim:</b> getting a model of pPDV100 for sequence alignment with purified pPDV089 plasmids from clones<br>
+<b>Time:</b> 14.00-16.00<br>
+<b>Method/Materials:</b> software Genious<br>
+<b>Results:</b> subsequent insertion of myc into reverse PCR primer (mdnA) caused frameshift, new primer ordered<br>

Team:Potsdam Bioware/Labjournal/July

From 2011.igem.org

Latest revision as of 21:15, 18 September 2011

Contents

28th Labday 2011-07-01

Agarose gel electrophoresis of PCR (mdnA for phage display, strategy 1)

purification of PCR product mdnA SfiI for phage display

Agarose gel electrophoresis of myc-tagged mdnA

29th Labday 2011-07-04

digestion of PCR product mdnA with restriction enzyme SfiI (strategy 1)

ligation of mdnA into pak100(strategy 1)

Amplification of mdnA for phage display (strategy 2)

Gel electrophoresis PCR product mdnA (strategy 2)

30th Labday 2011-07-06

digestion of PCR products mdnA, geneIII and vector pARW089 (strategy 2)

Gel extraction from agarose gel of digested fragments mdnA, geneIII and pARW089 (strategy 2)

31th Labday 2011-07-07

repeated digestion of pARW089 with NarI and AatII (strategy 2)

Gel electrophoresis of digested pARW089 vector(strategy 2)

ligation of mdnA and geneIII into pARW089(strategy 2)

32th Labday 2011-07-12

Transformation of ligation mdnA, geneIII, and pARW089 (2011-07-07) for phage display (strategy 2)

33th Labday 2011-07-13

Error prone PCR (varied MnCl2 concentration) of mdnA

34th Labday 2011-07-14

Agarose gel electrophoresis of error prone PCR of mdnA from 2011-07-13 and gel extraction

Digestion of error prone PCR of mdnA from 2011-07-13 and pARW089 and agarose gel electrophoresis

Overnight culture of PDV089

Repeated PCR of mdnA for phage display (strategy 1) repeated with new ordered reversed primer (with myc-tag) and geneIII

35th Labday 2011-07-15

Ligation of mutated mdnA into pARW089

Transformation of competent E. coli cells with mutated mdnA in pARW089

ligation of mdnA into pak100(strategy 1)

Transformation of ligation mdnA into PAK100 bla KDIR for phage display (strategy 1)

ligation of mdnA and geneIII for strategy 2

ligation of the fusion gene mdnA-geneIII into pARW089(strategy 2)

36th Labday 2011-07-16

Transformation of ligated pARW089 with mdnA-geneIII

Overnight culture of XL1 blue with mdnA in PAK100blaKDIR

37th Labday 2011-07-17

Miniprep of E. coli overnight culture containing PAK100blaKDIR

E. coli XL1 blue stock culture containing PAK100blaKDIR with mdnA (Phage Display-strategy 1)

overnight culture of E. coli Xl blue m. Ligation089jessi, u.Ligationvector089jessi,u.Ligationu.BandemdnAgeneIIIpARW089, mLigatiojnm.BandemdnAgeneIIIpARW089

38th Labday 2011-07-18

overnight culture of E. coli XL-1 blue containing mutated mndA in parW089

Dual-Tranformation of XL1-blu (E. coli) with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla

Test digestion of ligations for strategies 1 and 2

39th Labday 2011-07-19

Resistance test of the Dual-Transformation of XL1-blue (E. coli) with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla

Stock cultures of XL1-blue(E. coli)with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla and of XL1-blue(E. coli)with pTEV-SCS1 and of XL1-blue(E. coli)pJC354_ssTorA_NheI_CS-TEV_XhoI_bla

Design of Primers for BioBrick mdnA and mdn-Cluster

Miniprep of E. coli Xl blue containing mutated mndA in parW089

40th Labday 2011-07-20

Resistance test of the Dual-Transformation of XL1-blue (E. coli) with pTEV-SCS1 and pJC354_ssTorA_NheI_CS-TEV_XhoI_bla

Restriction enzyme digestion pARW089 and pARW071

overnight culture of E. coli Xl blue m. ligation mdnA and geneIII into pARW089

Repeated PCR of mdnA and gene III for phage display (strategy 1+2)

digestion of PCR products and vectors (strategy 1+2)

ligation of mdnA and geneIII into digested pARW089(strategy 2)

ligation of mdnA into pak100blaKDIR (stategy 1)

41th Labday 2011-07-21

Test digestion of ligation for strategy 2

Transformation of ligated pARW089 with mdnA-geneIII and ligated PAK100 bla KDIR with mdna

42th Labday 2011-07-22

Restriction enzyme digestion pARW089 and pARW071 AND trouble-shouting

43th Labday 2011-07-23

Restriction enzyme digestion pARW089 (different incubation times), plasmid purification and ligation with EP-PCR mdnA

creation of pPDV089 in Genious

sending pPDV089 from positive clone to GATC for sequencing

44th Labday 2011-07-24

Transform ligation from 42th Labday into E. coli XL blue 1 cells

45th Labday 2011-07-25

overnight culture of E. coli XL-1 blue containing mutated mndA in parW089

46th Labday 2011-07-26

Miniprep of E. coli Xl blue containing mutated mndA in parW089

Alignment of sequenced pPDV089 and model of pPDV089

Repeated PCR of mdnA and gene III for phage display (strategy 1+2)

digestion of PCR products and vectors (strategy 1+2)

gel electrophoresis of digested fragments and vectors

Repeated PCR gene III for phage display (strategy 2)