Team:Freiburg/Notebook/27 July

This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!

• < home >
• < team >
  • → members
  • → attributions
  • → freiburg
• < project >
  • → description
  • → results
  • → modeling
  • → notebook
  • → sample data
• < human practices >
  • → oath
  • → ethics
  • → safety
• < gallery >
• < sponsors >
• < to the forums >

Contents 1 Commons 1.1 Gel 2 green light receptor 2.1 Results of Sequencing 2.2 Quickchange PCR of CcaS and CcaR 2.3 Protocol 3 blue light receptor 3.1 PCR of Lov-tap and Not-gate 4 Precipitator

Commons

Gel

Investigators: Sandra, Sophie

We loaded the PCR products of the vectors on a gel and measured the DNA concentration. DNA concentration were high and we diluted the vectors to get an endconcentration of 25ng/microl.

DNA-concentration measured with nanodrop:

sample	DNA concentration (ng/μl)
pSB1C3	132.1
pSB1K3	101.5
pSB1A3	83.9
pSB1T3	116.2

green light receptor

Results of Sequencing

Investigators:Julia

CcaS 4a2 seemed to miss about 20bp, CcaS 4b2 is correct.

Quickchange PCR of CcaS and CcaR

Investigators:Julia

in order to eliminate iGEM restriction sites of EcoRI within CcaS and CcaR, we designed mutagenesis primer and performed a quickchange PCR.

Protocol

total reaction volume is 50µl:

32,5 µl H2O
10 µl Phusion Buffer 5x
2,5 µl Primer up
2,5 µl Primer dw
1 µl DNA template, concentration has to be 5ng/µl
0,5 µl Phusion

Primer for CcaR:
ccaR_mut1_up: CCtcactaATGAGgATcCTTTTAGTGGAGG
ccaR_mut1_dw:CCTCCACTAAAAGgATcCTCATtagtgaGG

Primer for CcaS:
ccaS_mut1_up:CCGGGCCGAGgATcCTCTATGTCAATG
ccaS_mut1_dw:CATTGACATAGAGgATcCTCGGCCCGG

Primer for CcaS:
ccaS_mut2_up:GGACCAAAAACgAGTCGCACTGAATTAG
ccaS_mut2_dw:CTAATTCAGTGCGACTcGTTTTTGGTCC

blue light receptor

PCR of Lov-tap and Not-gate

Investigators: Sandra, Sophie

We repeated the PCR again, with higher temperatures this time. After PCR we digested with DpnI 37°C for 1 hour and then purified the DNA with PCR purifictaion kit. Afterwards we measured the DNA concentration and loaded 2 microl to a gel.

PCR

Name:Sandra, Sophie	Date: 27.07.11
Continue from Experiment: NOT-Gate and LovTAP with Gibson-overhangs (now we use a little bit higher temperature for primer annealing) -(Date): 25.07.11 (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	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: 58°C and then after 10 cycles 68°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?

DNA-concentration measured with nanodrop:

sample	DNA-concentration (ng/μl)
S35	75.1
S45	45.1

Precipitator

Ruediger 27.07 picked colonies from yesterdays Trafo into medium with 2μl/ml Tet

S39-P18 S39-P19 S39-P20

S43-P18 S43-P19 S43-P20

overnight in 37°C