Team:Freiburg/Notebook/1 August

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Contents 1 green light receptor 1.1 Qickchange PCR of CcaS 2 blue light receptor 2.1 PCR 3 red light receptor 3.1 3A-assembly with pcyA and the terminator BBa_1006 3.1.1 1.Digestion: 3.1.2 2.Ligation 3.1.3 3.Transformation 4 Lysis cassette 4.1 Phage Lysis Cassette (K124017) + RBS (B0034) 5 Precipitator

green light receptor

Qickchange PCR of CcaS

Investigators:Julia
repeating experiment from___, because we had no positive transformation, probably chosen wrong annealing temperature before.

blue light receptor

PCR

Investigators: Sophie

As our last PCRs of the LovTAP with the Gibson overhangs didn't work well, we now try a PCR with primers without overhangs for Gibson assembly.

Name: Sophie	Date: 01.08.11
Continue from Experiment: PCR (Date): 27.07.11 (Name): Sandra, Sophie
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	P35
2.5µl	Primer dw	P36
1µl	dNTPs	of Template DNA
1µl	DNA-Template	M35 (LovTAP)
0.5 µl	Phusion (add in the end)

What program do you use?

LovTAP ohne Ueberhaenge

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?

Labelled: M35 short; stored in Gibson Stuff box.

red light receptor

3A-assembly with pcyA and the terminator BBa_1006

Investigators:Julia

1.Digestion:

terminator (S21a), 26µl of the miniprep+ 12,1 µl H2O, cut with XbaI&PstI
pcyA (part BBa_I15009), 3 µl of miniprep + 35µl H2O, cut with EcoRI&SpeI
Vector-backbone (10 µl) pSB1T3, cut with EcoRI,PstI & DpnI

to each reation 5µl BSA(10x) and 5µl NEB buffer4 were added, digested at 37°C for two hours, inactivated at 80°C for 20min.

2.Ligation

2µl of digested pcyA,terminator and vector were added to 11µl H2O,2µl ligase buffer and 1µl T4 ligase.
Incubated at room temperature for 35 min, inactivated at 80°C for 20 min.

3.Transformation

Lysis cassette

Phage Lysis Cassette (K124017) + RBS (B0034)

Investigators: Theo

the absence of a RBS (ribosome binding site) between the lamba cI regulated Promotor (R0051 of K098995) and the holin ATG of K124017

Precipitator

PCR

Name: Sophie	Date: 01.08.11
Continue from Experiment: replay of PCR 19.07.11 (Date) 19.07.11 (Name) Ruediger
Project Name: GFP Pbd

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	P28
2.5µl	Primer dw	P18 / P19/ P20
1µl	dNTPs	of Template DNA
1µl	DNA-Template	S 14 (GFP)
0.5 µl	Phusion (add in the end)

What program do you use?

Ruediger PCR (modified by Tobi and me. First 10 cycles with 55°C, next cycles with 62°C annealing temperature.

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?