Team:Freiburg/Notebook/25 July

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Contents 1 Commons 2 green light receptor 2.1 Testdigest: Ligation of CcaS into pSB1K3 3 blue light receptor 3.1 Gibson Not-Gate:Transformation 4 Precipitator

Commons

PCR

Name: Sophie	Date: 25.07.11
Continue from Experiment (Date) (Name): Commons
Project Name: more linearized backbones (4 different vectors)

PCR-Mixture for one Reaction:

For a 50 µl reaction use

32,5µl	H20	Name
10µl	5x Phusion Buffer	of Primer
2.5µl	Primer fw	SB-prep-3P-1
2.5µl	Primer dw	SB-prep-2Ea
1µl	dNTPs	of Template DNA
1µl	DNA-Template	PSB 1 A3 PSB 1 C3 PSB 1 K3 PSB 1 T3
0.5 µl	Phusion (add in the end)

What program do you use?

1. 2Min 94°C
2. 30s 94°C
3. 30s 55°C
4. 3min 72°C
5. 10min 72°C

step 2,3 and 4 in 35 cycles

Labeled PSB 1 A3, PSB 1 C3, PSB 1 K3 and PSB 1 T3 stored in -20°C in last drawer

green light receptor

Testdigest: Ligation of CcaS into pSB1K3

Investigators:JULIA

Testdigest

Continue from Experiment (Date 22.07) Ligation of CcaS into pSB1K3

Project Name: Green light receptor

For one reaction you need For Mastermix: 35 samples+2extra

4μl	H2O	178μl	H2O
1μl	Buffer, NEB4	37μl	Buffer, NEB4
1μl	BSA (10x)	37μl	BSA (10x)
0,5 μl	Enzym 1	7μl	EcoRI
0,5 μl	Enzym 2
3 μl	DNA

10 μl total volume

Give 3 μl of DNA in an eppi and add 7μl of the mastermix.

Incubate for about 1h at 37°C.

Add 1 μl Loading dye buffer and load the gel.

Picture of 1% gel:

result:4a2,4b2,4b3,4b4,4b5 should have the right insert. Send 4a2 and 4b2 to GATC for sequencing.

blue light receptor

Gibson Not-Gate:Transformation

Investigators: Sophie

Name: Sophie	Date: 25.07.11
Continue from Date Name Experiment
Project Name: Blue light (NOT-Gate)

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.

We need a NOT Gate for our Blue light system. In this experiment I transformed E.coli with the Part Bba_Q04400 from the iGEM distribution kit. The vector is pSBK3 with Kanamycin resistance. The strain ist named S 45 and will be plated out. The plates will be stored in the incubator.

PCR

Name: Sophie	Date: 25.07.11
Continue from Experiment: new experiment. We need NOT-Gate and LovTAP with Gibson-overhangs, so 2 PCRs are made
Project Name: Blue Light

PCR-Mixture for one Reaction:

For a 50 µl reaction use

32,5µl	H20	Name
10µl	5x Phusion Buffer	of Primer:
2.5µl	Primer fw	NOT_G_up LOV_G_up
2.5µl	Primer dw	NOT_G_dw LOV_G_dw
1µl	dNTPs	of Template DNA
1µl	DNA-Template	S35 (BBa_K322999) S45 (BBa_Q04400)
0.5 µl	Phusion (add in the end)

What program do you use?

"57°C auf 70°C" (first annealing temperature:55°c, after 10 cycles 65°c)

To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.

How did you label the PCR-Product, where is it stored and what do you do next?

I labelled it with a heart and a star and stored it in the minipreps 3 box.

I will do Gibson assemblz of the two parts next.

DNA-concentration measured with nanodrop:

sample	DNA-concentration (ng/μl)
S35	75.3
S45	27.2

We will repeat the PCr, because of the bad result of S35. We will do a PCR with higher temperature.

Precipitator

Digestion

Name: Ruediger	Date: 25.07
Continue from Date Name Experiment
Project Name:

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

Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:

Sample Name	DNA concentration (μg/μl)
P20 GFP	132
P19 GFP	145
P18 GFP	107
S39 PR	90
S43 PR	40
Tet Vector	25

Components	Vector (μl)	S39 Insert1: PR	S43 Insert1: PR	P18 Insert2: GFP+Pbd	P19 Insert2: GFP+Pbd	P20 Insert2: GFP+Pbd
DNA (500ng)	20	5.5	12.5	4.7	3.5	3.8
BSA (10x) (5μl)
NEB4 Buffer (5μl)
Enzyme 1 (1μl)	E	E	E	X	X	X
Enzyme 2 (1μl)	P	S	S	P	P	P
H2O (38 μl- DNA)	18	32.5	25.5	33.3	34.5	34.2
In total 50 μl

Documentation:

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

3A Assembly of linearized empty Tet Vector Psb1T3, PR Parts S39, S43 (CM resistance) and GFP Pbd from PCR with P18,19,20 I did not digest linearized PsB1T3 with Dpn.

Run a gel to verify if the part is cut out.