Team:Freiburg/Notebook/26 July

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Contents 1 blue light receptor 1.1 Gibson-Assembly 2 Precipitator 3 Lysis cassette 3.1 Sequencing of Quickchange-modified Lysis Repressor Part (modified K098995) 3.2 3A Assembly (K098995 + K124017)

blue light receptor

Gibson-Assembly

Investigators: Sandra, Sophie

Planned Gibson-Assembly for Lov-tap and Not-Gate. Need to order NAD. We will start an thursday.

Precipitator

Ligation

Name: Ruediger	Date: 26.07
Continue from Date Name Experiment Digestion 25.07 Ruediger
Project Name:GFPPbd

Procedure

PCR tube:

total volume 20 μl

1. add H₂O (17 μl -X-Y-Z)
2. add 2 μl Ligase Buffer 10x
3. add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
4. add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
5. Add 1 μl T4-DNA Ligase
6. Incubate 10-30 min at room temperature
7. heat for 20 minutes at 80°C
8. store at -20°C or directly proceed to transformation

Part name	Volume (μl)	Part name	Volume (μl)
S39	2	S43	2
P18	1,8	P18	1,8
Psb1T3	2	Psb1T3	2
S39	2	S43	2
P19	1,8	P19	1,8
Psb1T3	2	Psb1T3	2
S39	2	S43	2
P20	1,8	P20	1,8
Psb1T3	2	Psb1T3	2

Added 12,2 μl to every sample

Documentation:

Why are you doing this experiment? Where are your parts stored? Name the parts for ligation etc.

See digestion yesterday 3A assembly combination as shown in table.

Transformation

Name: RT	Date: 26.07
Continue from Date Name Experiment Ligation 26.07 Ruediger
Project Name: GFPPbd

Procedure

1. take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
2. thaw cells on ice 20 minutes
3. pipette 50 μl cells and 2 μl DNA into eppi still on ice!
4. Incubate for 30 minutes on ice
5. Heat at 42°C for 60 sec
6. Incubate on ice for 5 minutes
7. Add 200 μl LB Broth
8. Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
9. Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance

Documentation:

Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.

Had only 6 tet plates -> put 100 μl of each sample on each plate Incubator overnight for 30°C S39-P18 S39-P19 S39-P20 S43-P18 S43-P19 S43-P20

Lysis cassette

Sequencing of Quickchange-modified Lysis Repressor Part (modified K098995)

Right-click to download the annotated ape (.gb) file

3A Assembly (K098995 + K124017)

Digestion of 3A Assembly

Name: Theo	Date: 26.07.2011
Continue from Experiment : Quickchange V.3
Project Name: Lysis Cassette V.2 (3A assembly of quickchange-modified K098995 with Lysis genes K124017)

Procedure

1. add H₂O (38μl-DNA )
2. 5 μl NEB4 buffer (stored at iGEM’s, -20°C)
3. 5 μl 10x BSA (used 1:10 diluted sample stored at iGEM’s, -20°C)
4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
5. 1 μl restriction enzymes (stored at iGEM’s, -20°C)
6. heat for 1-2 hours 37°C (6 hours if time)
7. heat for 20 minutes 80°C (inactivation of enzymes)
8. keep at 4°C if you cannot continue

Restriction enzymes you need to cut the vector, insert1 and insert 2:

Components	Vector (μl)		Insert1 -K098995- and 2 - K124017-(μl)
DNA (500ng)		10		5	7
BSA (10x) (5μl)
NEB4 Buffer (5μl)
Enzyme 1 (1μl)		EcoRI + DpnI		EcoRI	XbaI
Enzyme 2 (1μl)		PstI		SpeI	PstI
H2O (38 μl- DNA)		27		33	31
In total 50 μl

Documentation:

Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.

3A assembly of quickchange-modified K098995 with Lysis genes K124017. Quickchange-modified K098995: 132,8ng/µl (Amp) Lysis genes K124017: (Amp+Kan) *Parts were run on the gel and verified that the inserts were cut, image accidentaly not saved* Vector: pSB1T3 + DpnI Digestion Stored in Theos Box

Ligation

Name: Theo	Date: 26.07.2011
Continue from: 26.07.2011 Lysis Cassette V.2 Digestion
Project Name: Lysis Cassette V.2 (3A assembly of quickchange-modified K098995 with Lysis genes K124017)

Procedure

PCR tube:

total volume 20 μl

1. add H₂O (17 μl -X-Y-Z)
2. add 2 μl Ligase Buffer 10x
3. add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
4. add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
5. Add 1 μl T4-DNA Ligase
6. Incubate 10-30 min at room temperature
7. heat for 20 minutes at 80°C
8. store at -20°C or directly proceed to transformation

	Name of part	Ratio Insert:Vector = 3:1 or 1:1	Volume (μl)
X insert 1	Modif. K098995	3:1 (1,5*3)	4,5
Y insert 2	K124017	3:1 (2*3)	6
Z vector	pSB1T3	1:3 (1)	1
H2O			5,5

Documentation:

Why are you doing this experiment? Where are your parts stored? Name the parts for ligation etc.

Ligation step of 3A assembly. How to calculate ratios --> e.g. for K124017 Length of pSB1T3= ca 2200bp Length of K124017+Vector= ca 4500bp 4500/2200= ca 2 So 2*3 µl (since 3:1 is needed) = 6 µl

Transformation

Name: Theo	Date:
Continue from : 26.07.2011 Lysis Cassette V.2 Ligation
Project Name: Lysis Cassette V.2 (3A assembly of quickchange-modified K098995 with Lysis genes K124017)

Procedure

1. take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
2. thaw cells on ice 20 minutes
3. pipette 50 μl cells and 2 μl DNA into eppi still on ice!
4. Incubate for 30 minutes on ice
5. Heat at 42°C for 60 sec
6. Incubate on ice for 5 minutes
7. Add 200 μl LB Broth
8. Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
9. Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance

Documentation:

Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.

New Composite Part in pSB1T3 Vector with quickchange-modified K098995 and Lysis genes K124017