Team:TU Munich/lab/notebook/part1

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Cloning Part I

People: Wolfgang, Tobi, Nico, Katharina, Flo


11-05-2011

Other Work

Parts requested from the registry:

Part Team Library Plate Well Plasmid Resistance
BBa_K238013iGEM09_KULeuven 2009 Submissions Shipment: 00413 1 pSB1A2 A
BBa_K228000 iGEM09_PKU_Beijing 2009 Submissions Shipment: 00399 1A pSB1A2 A
BB a_K322127 iGEM10_Edinburgh Submissions Shipment: 00694 6 pSB1C3 C
BBa_K228823 iGEM09_PKU_Beijing 2009 Submissions Shipment: 00401 11B pSB4K5 K
Bba_K322119 iGEM10_Edinburgh unknown unknown unknown unknown
Bba_K322115 iGEM10_Edinburgh unknown unknown unknownunknown

12-05-2011

Autoclave broken

Should be working next week again (Andrea is going to tell us...).

-> no agar plate/medium preparation possible right now.

But all other necessary stuff seems to be available (for electroporation, competent cells, miniprep-kit, DH5alpha strain, BL21 strain)

19-05-2011

Autoclave working

Other Work

Preparing Agar-LB Plates with Amp and Kan


20-05-2011

Cloning

Transformation

Transformation of all red-light sensor parts separately. DH5alpha cells were transformed using electroporation (V=1600V) with following constructs:

Part alias : Part Name - Plasmid - Year of Distribution, Plate Number

iGEM R1 : R0082 pSB1A3 2010, P1

iGEM R2 : I732017 pSB1A2 2010, P2

iGEM R3 : I15010 pSB2K3 2010, P3

iGEM R4 : K098010 pSB4C5 2010, P3

Each electroporation was performed with 1,5µl bio-brick plasmid (each disolved in 10ul ddH2O before) and 40µl competent DH5alpha cells according to the protocol kindly provided by Andrea Mueckl

Afterwards cells were incubated in 1ml SOC medium for 1,5 h at 37°C, 200rpm and subsequently plated on antibiotic containing LB-agar plates (50µl, 100µl, 200µl) and incubated at 37°C over night and then kept at 4°C.


23-05-2011

Results

Transformation of all red sensor parts failed

Cloning

Transformation

Transformation of one red part sensor (iGEM R1) with slightly modified protocol: electroporation performed at 1500V with 1 µl Plasmid (part previously disolved in 10ul ddH2O)and 40ul competent DH5alpha. Otherwise same procedure as before.

Other Work

Making electrocompetent cells according to Andrea's Mueckl protocol


24-05-2011

Results

Transformation of R1 resulted in 3 colonies

Other Work

Preparing over-night culture (20 mL) of DH5alpha cells in Luria-Media for electrocompetent cells

Preparing over-night culture of one clone picked from the successful second transformation

Asked J. Winter for heatresistant e.coli. We should get them tomorrow after lunch on a plate


25-05-2011

Results

Clone turned out as the right one.

J. Winter wants us to come back tomorrow, since bacteria seem to be contaminated

Cloning

MiniPrep

Testing clone: Mini-Prep using Zymoresearch DNA Kit, Digestion with EcoRI and PstI in NEB-Buffer 4, Gel (expected fragments 0,1 kbp (=R1) and 2,0 kbp (=pSB1A2))

Other Work

Continue making cells electrocompetent


26-05-2011

Results

Again: J. Winter wants us to come back next day

Cloning

Transformation

Transformation of

Part alias : Part Name - Plasmid - Year of Distribution, Plate Number

iGEM R2 : I732017 pSB1A2 2010, P2

iGEM R3 : I15010 pSB2K3 2010, P3

iGEM R4 : K098010 pSB4C5 2010, P3

using 1510V and 1ul DNA for transformation. DNA was just added to the cells and gently mixed by stirring it with the pipette tip. (Andrea Meyer's protocol) Of the 1ml SOC culture, 50ul were spread out directly on agar plates. Rest was spun down (2000rpm, 1min) and pellet was resuspended in 100-150ul. Subsequently, 50ul were spread out.


27-05-2011

Results

successful clones from transformed cells with iGEM R2 and iGEM R4, no clones from iGEM R3 at all

Again: J. Winter wants us to come back next week, still contamination in the culture

Cloning

Transformation

Transformation of:

Part alias : Part Name - Plasmid - Year of Distribution, Plate Number

iGEM R3 : I15010 pSB2K3 2011, P3

iGEM tRNA : K228001, pSB1A2 2011, P4

iGEM Term : B0015 pSB1AK3 2011, P1

iGEM RBS : J44001 pSB1A2 2011, P1

iGEM T7 : I712074 pSB1AK8 2011, P1

since the transformation with iGEM R3 did not work, we increased the plasmid amount to 2 µl

afterwards the procedure was performed as mentioned above and the cells were incubated over night (15 h) at 37 °C and cooled until monday at 4 °C


30-05-2011

Results

Successful clones from transformed cells with iGEM tRNA, iGEM Term, iGEM RBS, iGEM T7

Finally, we received BM28 cells from J. Winter (there is an existing paper in JBC from her about the bacteria)

Cloning

Transformation

Transformation of R3 once more using BL21 and inducing pSB2K3 plasmid using 1mM IPTG in SOC-Medium

Other Work

Over-night culture of clones from iGEM tRNA, iGEM Term, iGEM RBS, iGEM T7, iGEM R2 und iGEM R4


31-05-2011

Results

Transformation of iGEM R3 did not work

Plasmid Concentrations:

- iGEM T7: 62 ng/µl

- iGEM R4: 82,5 ng/µl

- iGEM tRNA: 98 ng/µl

- iGEM R2: 94 ng/µl

- iGEM Term: 141 ng/µl

- iGEM RBS: 108 ng/µl

- iGEM R1: 59 ng/µl

Cloning

MiniPrep

Plasmid Isolation (MiniPrep) from all over night cultures


01-06-2011

Results

Gel run inconclusive, next time longer run with higher voltage - only 1/3 of the lane was used by the peptides. Very bad resolution - dying step worked properly

Cloning

Digestion

- Restriction digestion with EcoRI and PstI in NEB-Buffer 4

- Gel run with digestion: 100 V, 1 h 10 Min, 1 % agarose

- CyberGold Staining: 2 µl on shaking plate for 30 mins @ RT

Transformation

Transformation of DH5alpha with:

iGEM R3 (see above) on A, C and K resistance plates

iGEM High Copy Plasmid, Plate 1 2011 1G, psB1A3 with insert: BBa_J04450, Resistance: A

iGEM Low Copy Plasmid, Plate 1 2011 3C, psB3C5 with insert: BBa_J04450, Resistance: C

plated on agar plates over night


03-06-2011

Results

Gel run again inconclusive, during run loss of electrical power because of some blackout

transformed cells grew very well over night, few clones of iGEM R3 on canamycine resistance plate found, probably contamination because incubator did not cool down to 4 °C over thursday

transformed plasmid-cells grew also very good - now featured in red

Cloning

Digestion

Gel run with digestion: 90 V, 1 h 30 Min, 1 % agarose

CyberGold Staining: 2 µl on shaking plate for 30 mins @ RT


06-06-2011

Results

Gel still inconclusive, may be too less material, since no bands around 100 bp could be detected

Cloning

Digestion

Gel run with newly digested and undigested plasmid as control: 100 V, 1 h 30 Min, 1 % agarose

Transformation

Transformation of R3 in E. coli D1210

Other Work

Over-night culture of:

iGEM High Copy Plasmid, Plate 1 2011 1G, psB1A3 with insert: BBa_J04450, Resistance: A

iGEM Low Copy Plasmid, Plate 1 2011 3C, psB3C5 with insert: BBa_J04450, Resistance: C


07-06-2011

Results

Transformation of R3 finally worked using E. coli D1210

Over night culture of iGEM Low Copy Plasmid, Plate 1 2011 3C, psB3C5 with insert: BBa_J04450, Resistance: C resulted in 101 ng/µl in a total volume of 100 µl.

We've ran out of DNA-Plasmid Preparation Kit...no preparation possible during next days!

Gel still inconclusive

Cloning

Digestion

2 % Agarose with Ethidiumbromid, 90V, 1.5 h

Digestion of 2 µg Plasmid DNA with high fidelity 1.: EcoRI and PstI enzymes and 2.: just with PstI



08-06-2011

Results

gel: R1, R2, R4 tRNA und Term seem ok; T7 promotor (46bp) and RBS (15bp) could not be detected ---> PAGE necessary!

Cloning

Digestion

1.5 % Agarose with Sybr Gold (Dilution: 1 µl/10ml), 90V, 1.0 h



09-06-2011

Results

gel: RBS, T7 digestion -> PAGE not successful;

DNA Conc. after MiniPrep:

BBa_K238013 on pSB1A2: 49ng/ul

BBa_K228000 on pSB1A2: 204 ng/ul

BBa_K322127 on pSB1C3: 168 ng/ul

Cloning

MiniPrep

MiniPrep of BBa_K238013 BBa_K228000 BBa_K322127

Other Work

Over Night Culture of R3 clone

Testing

SDS Page

10% PAGE of RBS and T7



10-06-2011

Results

gel:

DNA Conc. after MiniPrep:

R3 188 ng/ul


Cloning

MiniPrep

MiniPrep of R3

Comparison of "new" (ordered) enzymes vs. "old" ones



14-06-2011

Results

gel: File:140611 gel beschriftet.pdf

all enzymes do work apparently


Other Work

Comparison of "new" (ordered) enzymes vs. "old" ones - running the gel (1% agarose, 100-150ml Gel) (Cut R4, R2)


16-06-2011

Cloning

Cloning of the red light sensor with parts R1,R2,R4 (see cloning draft)

ONC of part containing colonies in 5ml liquid culture



17-06-2011

Results

NO COLONIES of first ligation ->problem: wrong enzymes used! (forgot to cut the linear fragment in the second step with pstI)

gel: inconclusive

Cloning

Ligation

Agarose gel of ligation products to evaluate ligation problems

MiniPrep

MiniPreps of Parts



20-06-2011

Results

Gel of samples collected during the red light sensor assembly: FILE


Cloning

-Cloning of the red light sensor with parts R1,R2,R4 (see cloning draft)

Other Work

-Backup Plates of existing clones



21-06-2011

Results

-Some strange looking colonies of red light sensor transformation

Cloning

-ONC of part containing colonies and red light sensor construct colonies in 5ml liquid culture

Cloning

-Cloning of the blue sensor (only step1) with parts K228000, RBS (see cloning draft)



22-06-2011

Results

-nice colonies of red light sensor transformation

Cloning

-Cloning of the blue sensor (step2) with parts: Y44001 (RBS) + K22800 = A1 and B 0015 (Term)

-Cloning of the red light sensor with lacZ (R2)

Digestion

- gel run of all products (110 V, 1%, 1h): lanes: 2log ladder, RBS undigested (=ud), RBS digested (dig), K22800 ud, K22800 dig, A1 ud, A1 dig, Term ud, Term dig, L1 ud, L1 dig, L2 ud, L2 dig, R2 ud, R2 dig 6 µl each lane gel: 220611 digest control 2.jpg

Transformation

-Transformation of both ligations (red and blue light) and incubation on plates over night:

- blue light on amp and kan

- red light I on amp plate not protected from light, lightning during electroporation

- red light II on amp 1 plate protected from light, 1 plate not protected from light

MiniPrep

- plasmid isolation of all red light sensor parts and the successfully transformed colonies from 21.06.11 concentrations in ng/µl

R1 93.5 R2 108.0 R3 37.0 R4 263.0 RBS 47.5 tRNA 154.0 Term 65.0 Ligation of transformed colony (21.6.11) I 95.9 Ligation of transformed colony (21.6.11) II 63.5 T7 106.0 high copy plasmid 142.0 low copy plasmid 66.5



24-06-2011

Other Work

New agarose plates



27-06-2011

Other Work

Design of new cloning strategy together with instructors



28-06-2011

Cloning

Digestion (EcoRIxPstI) and ligation of K238013, tRNA in pSB3C5, blue light construct (j44001-K22800-B0015), Reporter construct (T7-R2)



29-06-2011

Results

Bad gel

Cloning

Digestion (EcoRIxPstI) of K238013, tRNA in pSB3C5, blue light construct (j44001-K22800-B0015), Reporter construct (T7-R2)



30-06-2011

Results

Agarose Gel of K238013 (Insert: 86bp), tRNA in pSB3C5 (Insert: 136bp), blue light construct (j44001-K22800-B0015) (Insert: 3,2kb), Reporter construct (T7-R2) (Insert: 3,1kb)

File:300611 analyse gel.pdf

In T7-R2 only T7 and in blue light construct only B0015? Maybe there is also the right band. Do our ligations work? (see cloning road maps...)

Cloning

Digestion

Digestion (ExP) of B0015, pSB1A3 (not needed), pSB3C5

Agarose Gel of K238013, tRNA in pSB3C5, blue light construct (j44001-K22800-B0015), Reporter construct (T7-R2)



01-07-2011

Cloning

Amplification

PCR to amplify certain parts out of the red light sensor of edinburgh: K322123, K322124, ompR:

PCR-Program:

1) Initial Denaturation: 96°C, 4'

2) Touchdown (8x)

   Denat.: 96°C, 30 s
   Annealing: 65°C -> 61,5°C; -0,5°C/cycle, 20 s
   Extension: 72°C, 4'

3) Constant Temp. (22x)

   Denat.: 96°C, 30 s
   Annealing: 61°C, 20 s
   Extension: 72°C, 4'

4) Final Extension: 72°C, 8'

5) Hold: 4°C, oo

PCR-mix (8x) (Taq-PCR-Kit; NEB):

water (autoclaved): 326 µl

standard buffer (10x): 40 µl

dNTPs (10mM): 8 µl

Taq-Pol: 2 µl

47 µl per reaction + 1 µl of each primer + 1 µl DNA -> 50 µl reaction volume

samples:

1) negative control: primer (10 µM) + water

2) DNA: 1,5 ng; primer (10 µM)

3) DNA: 15 ng; primer (10 µM)

4) DNA: 1,5 ng; primer (5 µM)

5) DNA: 15 ng; primer (5 µM)

6) DNA: 1,5 ng; primer (1 µM)

7) DNA: 15 ng; primer (1 µM)

Other Work

Agarose Gel of ladder, R2, T7, Pol, lac-Pol(R2-Pol), T7-R2, Term. low copy 1:1, Term low copy 3:1, ladder 2 runs with different conditions: 120 V, 400 mA, 1.5 h<-> 80 V, 120 mA, 1.5h


Making of electrocompetent cells of DH5alpha and BM28



04-07-2011

Results

040711 Plasmid2 analyse inverse.jpg


Cloning

Agarose

Agarose Gel of ladder, R2, T7, Pol, lac-Pol(R2-Pol) ligation, T7-R2 ligation

analytical agarose gelelectrophoresis of the PCR samples from 01-07-11: (1%, 1x TBE, 120 V, 400 mA, 90 min)

10 µl PCR sample + 2 µl loading buffer -> 10 µl per well

lanes:

1.) 2-log

2) negative control: primer (10 µM) + water

3) DNA: 1,5 ng; primer (10 µM)

4) DNA: 15 ng; primer (10 µM)

5) DNA: 1,5 ng; primer (5 µM)

6) DNA: 15 ng; primer (5 µM)

7) DNA: 1,5 ng; primer (1 µM)

8) DNA: 15 ng; primer (1 µM)

9) 2-log

040711-PCR-redlight-1.jpg



05-07-2011

Cloning

Amplification

repetition of PCR to amplify certain parts out of the red light sensor of edinburgh: K322123, K322124, ompR:

Phusion High-Fidelity PCR-Kit (NEB/Finnzymes)

PCR-Program:

1) Initial Denaturation: 98°C, 4'

2) 30 cycles of:

   Denat.: 98°C, 30 s
   Annealing: 60°C, 15 s
   Extension: 72°C, 2´ 30 s

3) Final extension: 72°C, 8'

4) Hold: 4°C, oo

Samples

PCR-samples were prepared with HF- and GC-buffer, respectively. End volume of the reaction setups was 50 ul.

DMSO-concentration was 100%; primer concentration was 10 uM, which equals an end conc. of 500 nM (2.5ul) or 200 nM (1ul).

DNA-template [150 ng/ul] was diluted 1:100 to a conc. of 1.5 ng/ul -> 1 ul of dilution was used per reaction.

Samples were prepared as follows (same setup for HF- and GC-buffer):

060711-PCR-Ansatz.jpg


06-07-2011

Results

Gel Verdau RBS-K228000.jpg


HF-buffer samples gel analysis:

060711-PCR-Testgel-HF-invers-beschriftet.jpg -> PCR seems to have been successful, amplicon is at about 4kb!

QC-buffer samples gel analysis (before and after cut-out):


QC-buffer-samples-PCR-vorher+nachher.jpg -> PCR also successful, cut-out of 4 bands, DNA prep and pooling samples

Cloning

Digestion

Digestion of J44001, K228000

Preparative gel run with the digested parts (1% agarose, 1xTAE, 120 V 400 mA, 1.5 h)

Isolation of the bands out of the gel

Preparation with a gel isolation kit (freeze ´n squeeze, bio-rad)

work-up of PCR-run (05-07-2011): gel analysis (1% agarose, 1xTBE, 120 V, 400 mA, 1.5 h) of HF-buffer-samples

gel analysis (1% agarose, 1xTBE, 120 V, 400 mA, 1.5 h) of GC-buffer-samples

isolation of right bands out of the GC-gel

DNA preparation with a gel isolation kit (freeze ´n squeeze, bio-rad)

restriction digest with EcoRI and SpeI

MiniPrep

mini-prep of culture (4 ml) from DH5alpha (05-07-2011) (qiagen qiaprep spin miniprep kit) -> 82 ng/µl in 50 µl elution buffer

restriction digest of 0.5 µg DNA with EcoRI and PstI

gel analysis of restriction digest (1% agarose, 1xTAE, 120 V, 400 mA, 1.5 h)



07-07-2011

Cloning

Ligation

Ligation (total volume: 50 µl; 1.5 µg of each DNA sample; 5 µl T4-ligase buffer (10x); 1 µl Quick-Ligase; add to 50 µl with nf H20; 60`@ 37°C)

Ligation of:

1. K228000 + RBS

2. K322127 + supD-tRNA

3. lacZ + T7-Promotor


08-07-2011

Results

Ligations failed

080711 ligationen failed.jpg


Cloning

preparative gel for ligation from 07-07-11; 1% TAE, 8 wells, 120 V, 400 mA, 90';

30 µl DNA + 6 µl loading dye (6x) -> 36 µl, 35 µl loaded on gel

7,5 µl 2-log DNA ladder



11-07-2011

Cloning

Ethanol precipitation (1/10 of volume 3M NaAc (pH 5.8), 2.5 X EtOH (100%, icecold)) of:

- Backbone (T7-Promotorpart, cut with S, P): 200 µl

- RBS (cut with S, P): 70 µl

- K228000 (cut with X, P): 90 µl

- K322127 (PCR-product redlight w/o lacZ; cut with E, S): 180 µl

- lacZ (cut with X, P): 90 µl

Add NaAc and EtOH (100%), invert 5x, incubate 30' @ -80°C

centrifuge 30' @ 4°C, 10000 rcf

no visible pellet -> centrifuge again, 30' @ 4°C, 18000 rcf

discard supernatant

wash with EtOH (70%, icecold) -> centrifuge 5' @ 4°C, 18000 rcf

discard supernatant

dry pellet (30' @ RT)

resuspend in 10 µl nuclease-free H20 (at no time visible pellet. nanodrop measurements suggested no detectable amount of DNA)

Restriction

Restriction digest (total volume: 50 µl; Enzymes: 1 µl each; 5 µl NEB 4 buffer (10x); incubation: 1 h @ 37°C; inactivation: 20 ' @ 80 °C) of PCR samples 2 and 4 (HF-buffer) from 05-07-11:

DNA: 10 µl (sample 2: 1 µl primer [10 µM], -DMSO; sample 4: 1 µl primer [10 µM], +DMSO)

H20: 33 µl

Enzymes: E, S


Preparative agarosegel (1%, 1xTAE, 8 wells, 120 V, 400 mA, 90'):

35 µl restriction digest + 7 µl loading dye (6x) -> 42 µl; 40 µl loaded on gel

7 µl 2-log DNA ladder

lanes:

1.) 2-log

2.) PCR-sample 2 from 05-07-11

3.) PCR-sample 4 from 05-07-11

11711 pcr präp.jpg 11711 pcr präp danach.jpg


12-07-2011

Cloning

Digest

Restriction digest (total volume: 50 µl; DNA: 1,5 µg; Enzyme: 1 µl each; 5 µl NEB 4 buffer (10x); incubation: 1 h @ 37°C; inactivation: 20 ' @ 80 °C) of the following parts:

- lacZ: 94 ng/µl -> 16 µl; H20: 27 µl; Enzymes: X, P

- T7-Promotor: 106,6 ng/µl -> 14 µl; H20: 29 µl; Enzymes: S, P

- SupD-tRNA: 150 ng/µl -> 10 µl; H20: 33 µl; Enzymes: E, X (#1, #2: double preparation for validation of different subsequent DNA purification steps)

- K238013 (blue light promotor): 233 ng/µl -> 6,5 µl; H20: 36,5 µl; Enzymes: E, X

- B0015 (Term.): 141 ng/µl -> 10,5 µl; H20: 32,5 µl; Enzymes: E, S

- RBS: 108 ng/µl -> 14 µl; H20: 29 µl; Enzymes: S, P


preparative agarosegel (1%, 1xTAE, 8 wells, 120 V, 400 mA, 90'):

35 µl restriction digest + 7 µl loading dye (6x) -> 42 µl; 40 µl loaded on gel

7 µl 2-log DNA ladder

1) DNA ladder

2) lacZ

3) T7 Promotor

4) supD tRNA

5) K328013

6) RBS

120711 verdau 1 prozent gel-nachher.jpg


preparative agarosegel (2%, 1xTAE, 8 wells, 120 V, 400 mA, 90'):

35 µl restriction digest + 7 µl loading dye (6x) -> 42 µl; 40 µl loaded on gel

7 µl 2-log DNA ladder

0) DNA ladder (pipette tip fell off)

1) DNA ladder

2) B0015 (Term.)

120711 b0015 2 prozent gel.jpg 120711 b0015 2 prozent gel-nachher.jpg

Ligation

Ligation (total volume: 30 µl; 10 µl of each DNA sample; 3 µl T4-ligase buffer (10x); 1 µl Quick-Ligase; 6 µl nf H20; 30`@ 37°C)

Ligation of:

1. lacZ + T7-Promotor

2. K322127 (PCR-Product) + supD-tRNA

3. K238013 + B0015


13-07-2011

Cloning

MiniPrep

Preparation of 24 overnight-cultures (5 ml each) for miniprep

Picking

picking of 3 clones (labelled a, b and c) per positive (= visible colonies) plate:

plate 1: lacZ + T7-promotor AFTER purification -> no colonies visible; Kan-resistance

plate 2: lacZ + T7-promotor BEFORE purification -> colonies; Kan-resistance

plate 3: PCR-product (from K322127) + supD-tRNA AFTER purification -> colonies; Amp-resistance

plate 4: PCR-product (from K322127) + supD-tRNA BEFORE purification -> colonies; Amp-resistance

plate 5: K238013 + B0015 AFTER purification -> colonies; Amp-resistance

plate 6: K238013 + B0015 BEFORE purification -> colonies (half normal, half slightly red); Amp-resistance

plate 7: K228000 + RBS AFTER purification -> colonies; Amp-resistance

plate 8: K228000 + RBS BEFORE purification -> colonies; Amp-resistance

plate 9: PCR-product (from K322127) + supD-tRNA BEFORE purification [ligated with T4 ligase] -> colonies; Amp-resistance


picking occured with 200 µl pipette tip, thrown into 15 ml Falcon with 5 ml LB + antibiotic (1:1000)

shaking started at 18:00 p.m., 200 rpm, 37°C, o/n

Other Work

Analytical agarosegel [130711-ligation-control.tif] (1%, 1xTBE, 20 wells, 120 V, 400 mA, 90') for control of ligations from 12-07-11:

10 µl ligation sample + 2 µl loading dye (6x) -> 12 µl, 10 µl loaded on gel

5 µl 2-log DNA ladder

lanes:

1) 2-log

2) lacZ + T7-promotor AFTER purification

3) lacZ + T7-promotor BEFORE purification

4) PCR-product (from K322127) + supD-tRNA AFTER purification

5) PCR-product (from K322127) + supD-tRNA BEFORE purification

6) K238013 + B0015 AFTER purification

7) K238013 + B0015 BEFORE purification

8) K228000 + RBS AFTER purification

9) K228000 + RBS BEFORE purification

10) PCR-product (from K322127) + supD-tRNA BEFORE purification [ligated with T4 ligase]

11) 2-log

130711-ligation-control.jpg


14-07-2011

Results

Ligation worked for K228000 + RBS (all six samples 7 a-c and 8 a-c), K238013 + B0015 (6c), lacZ+Z7-Promotor (2b)

Ligation failed for K322127 + supD-tRNA

Cloning

MiniPrep

- miniprep (4 ml) of the 24 over-night cultures from 13-7-11:

2a-c: lacZ + T7-promotor BEFORE purification

3a-c: PCR-product (from K322127) + supD-tRNA AFTER purification

4a-c: PCR-product (from K322127) + supD-tRNA BEFORE purification

5a-c: K238013 + B0015 AFTER purification

6a-c: K238013 + B0015 BEFORE purification

7a-c: K228000 + RBS AFTER purification

8a-c: K228000 + RBS BEFORE purification

9a-c: PCR-product (from K322127) + supD-tRNA BEFORE purification [ligated with T4 ligase]


- measurement of the DNA concentration with nanodrop

2a: 25 ng/µl

2b: 215 ng/µl

2c: 9.5 ng/µl

3a: 85 ng/µl

3b: 53.5 ng/µl

3c: -

4a: 226 ng/µl

4b: 20 ng/µl

4c: 57.5 ng/µl

5a: 68.5 ng/µl

5b: -

5c: 20.5 ng/µl

6a: 360 ng/µl

6b: 143 ng/µl

6c: 305 ng/µl

7a: 443 ng/µl

7b: 218 ng/µl

7c: 204 ng/µl

8a: 228 ng/µl

8b: 268 ng/µl

8c: 218 ng/µl

9a: 38.5 ng/µl

9b: 16 ng/µl

9c: 16.5 ng/µl

Restriction

restriction digest of 19 plasmids from the miniprep (without 2c, 3c, 5b, 9b, 9c)

Restriction digest (total volume: 30 µl; DNA: 0,5µg; Enzyme (EcoR1 + Psi): 1 µl each; 3 µl NEB 4 buffer (10x); incubation: 50´@ 37°C)


Analytical agarosegel (1%, 1xTBE, 20 wells, 120 V, 400 mA, 50') for control of transformation success from 13-07-11:

10 µl sample + 2 µl loading dye (6x) -> 12 µl, 11 µl loaded on gel

5 µl 2-log DNA ladder

lanes:

0.) 2 log

1.) 2a -> lacZ + T7-promotor BEFORE purification

2.) 2b -> lacZ + T7-promotor BEFORE purification

3.) 3a -> PCR-product (from K322127) + supD-tRNA AFTER purification

4.) 3b -> PCR-product (from K322127) + supD-tRNA AFTER purification

5.) 4a -> PCR-product (from K322127) + supD-tRNA BEFORE purification

6.) 4b -> PCR-product (from K322127) + supD-tRNA BEFORE purification

7.) 4c -> PCR-product (from K322127) + supD-tRNA BEFORE purification

8.) 5a -> K238013 + B0015 AFTER purification

9.) 5c -> K238013 + B0015 AFTER purification

10.) 6a -> K238013 + B0015 BEFORE purification

11.) 6b -> K238013 + B0015 BEFORE purification

12.) 6c -> K238013 + B0015 BEFORE purification

13.) 7b -> K228000 + RBS AFTER purification

14.) 7a -> K228000 + RBS AFTER purification

15.) 7c -> K228000 + RBS AFTER purification

16.) 8a -> K228000 + RBS BEFORE purification

17.) 8b -> K228000 + RBS BEFORE purification

19.) 8c -> K228000 + RBS BEFORE purification

19.) 9a -> PCR-product (from K322127) + supD-tRNA BEFORE purification [ligated with T4 ligase]

14711 digest-after-miniprep.jpg



15-07-2011

Results

in all samples and plates failed the experiment. There was no useable DNA detected

Cloning

PCR

Colony PCR for the ligation of K322127 and supD-tRNA. Therefore 15 colonies of the plates 3,4 and 9 (from 13-7-11) were resuspended in 50 µl nuclease free water. The experimet failed

Analytical agarosegel (1%, 1xTBE, 20 wells, 120 V, 400 mA, 90') for control of lthe colony-PCR from 15-7-11: 10 µl ligation sample + 2 µl loading dye (6x) -> 12 µl, 10 µl loaded on gel 5 µl 2-log DNA ladder

plate 3:

lanes:

1. 2-log

2. positive control

3-17. colonies from plate 3

18. positive control

150711-Gel1-PCR-Platte3.jpg

plate 4:

lanes:

1. 2-log

2. negative control

3-17. samples from the colonies of plate 4

15711 colonyPCR-NK-platte4d-r.jpg


plate 9:

lanes:

1. 2-log

2-17. samples from colonies of plate 9

15711-colonyPCR platte9d-r.jpg



19-07-2011

Results

restriction digest worked

Cloning

Restriction

Restriction digest of K238013+B0015 with S, P and K228000 (T7-Polymerase) + RBS with X, P

Restriction digest (total volume: 30 µl; DNA: 1 µg; Enzyme 1 µl each; 3 µl NEB 4 buffer (10x); incubation: 1 h @ 37°C)

The used DNA-templates were 8a (14-7-11) and 6c (14-7-11)


preparative agarosegel (1%, 1xTAE, 8 wells, 120 V, 400 mA, 90'):

30 µl restriction digest + 6 µl loading dye (6x) -> 35 µl loaded on gel

lanes:

1.) 2 log- DNA ladder 7 µl

2.) K22800 + RBS

3.) K238013 + B0015

4.) K322127 from an earlier restriction digest

-> DNA extraction from gel via mi-Gel-extraction kit from metabiom

19711 verdau t7pol-RBS K238013-B0015 K322127.jpg 19711 verdau t7pol-RBS K238013-B0015 K322127 nachher.jpg



20-07-2011

results

ligation success inconclusive

Cloning

Ligation

ligation of K228000-RBS with K238013-B0015 and K322127 with subD-tRNA.

Ligation (total volume: 30 µl; 10 µl of each DNA sample; 3 µl T4-ligase buffer (10x); 1 µl Quick-Ligase; 6 µl nf H20; 30`@ 37°C)

Transformation

transformation (40 µl competent cells; 1 µl DNA; Electroporation at 1510 V; incubation in Soc-Medium for 1 h @ 37°C, 50 µl on Amp-agar plate)

analytical agarosegel (1%, 1xTBE, 8 wells, 120 V, 400 mA, 90')

10 µl DNA + 2 µl loading dye -> 10 µl on gel

Lanes:

1.) 7 µl 2-log DNA ladder

2.) K228000-RBS + K238013 - B0015 (upper band in preparative gel)

3.) K228000-RBS + K238013 - B0015 (lower band in preparative gel)

4.) K322127-tRNA

20711 ligation analytisch.jpg



21-07-2011

Cloning

Restriction

control restriction digest with the Enzymes E, P of 2b (=ligation product lacZ+T7 promotor), 6c (=ligation product K238013+B0015) and 8a (ligation product K228000+RBS).

Restriction digest (total volume: 30 µl; DNA: 0.5 µg; Enzyme 1 µl each (E, P); 3 µl NEB 4 buffer (10x); incubation: 1 h @ 37°C)

analytical gelelektrophoresis (1%, 1x TBE, 12 wells, 120 V, 400 mA, 90´)

lanes:

1.) 2b ( =ligation product lacZ+T7-Promotor)

3.) 6c (=ligation product K238013 B B0015)

5.) 8A (=ligation product K228000 + RBS)

6.) 2-log DNA ladder

21711 kontrollverdau 2b 6c 8a.jpg

Other Work

production of LB-Agar plates with ampicillin

- 5 ml overnight culture of the transformated cells from 20-7-11. From each transformation 3 colonies were picked and cultivated in 5 ml LB-medium with Ampicillin.

-> 10 a-c: ligation product K322127 + tRNA

-> 11 a-c: ligation product K228000-RBS + K238013+B0015

- 5ml overnight culture of 218 (pBADlacZ) with Ampicillin

- 5 ml overnight culture of BM28 with Kanamycin

-> both cultures are used for gelrite experiments


22-07-2011

Cloning

MiniPrep

miniprep of the 4 ml overnight cultures from 21-7-11 with metabion

10 a-c: ligation product K322127 + tRNA

11 a-c: ligation product K228000-RBS + K238013+B0015

- nanodrop measurement of the DNA concentration:

10 a: 62 ng/µl

10 b: 42 ng/µl

10 c: 38.5 ng/µl

11 a: 51 ng/µl

11 b: 47.5 ng/µl

11 c: 47.5 ng/µl

Restriction

control regristiction digest of 10 a-c (ligation product K322127 + tRNA) and 11 a-c (ligation product K228000-RBS + K238013+B0015) with E, P

Restriction digest (total volume: 30 µl; DNA: 0.3 µg; Enzyme 1 µl each (E, P); 3 µl NEB 4 buffer (10x); incubation: 1 h 10` @ 37°C, inactivation 20´@ 80°C)

analytical gelelectrophoresis (1%, TAE 1x, 12 wells, 120 V, 400 mA, 120`)

lanes:

1.) 2-log ( 1 µl 2-log, 1 µl loading buffer (6x), 10 µl water) -> 10 µl loaded on gel

2.) 10 a

3.) 10 b

4.) 10 c

5.) 11 a

6.) 11 b

7.) 11 c

110722 Gel Right 1.jpg