Difference between revisions of "IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/08/16"

From OpenWetWare
Jump to: navigation, search
(Ligation of pTet)
Line 6: Line 6:
 
| colspan="2"|
 
| colspan="2"|
 
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
 
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==Transformation results (from the day before)==
 
* pSG.008: 1 colony
 
* pSG.021: 4 colonies
 
* pSG.017, pSG.018: lots of colonies
 
* pSG.006: no colony
 
  
 
==Colony PCR==
 
==Colony PCR==

Revision as of 09:43, 17 August 2012

Owwnotebook icon.png Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>      </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Colony PCR

We did colony PCR for all the colonies of pSG.008 and pSG.021

Colony+50μL H2O:

  • 10μL for cell culture (in 10mL LB+antibiotic)
  • boil the rest for 5min, take 1μL for as template DNA

Protocol

H2O 36.5 uL
5x Phusion HF Buffer 10 uL
10 mM dNTPs 1 uL
Primer mix 1 uL
Template DNA 1 uL
Phusion DNA polumerase 0.5 uL

PCR Program

Cycle step Temperature (C) Time Cycles
Initial denaturation 98 30s 1
Denaturation 98 5-10s 30*
Annealing 56 10-30s 30*
Extension 72 1m 30*
Final extension 72 5-10m 1
4 hold 1

Ligation of pTet

We ligated pTet (annealing product) with pSG.002 (E,S), Kan resistant

Protocol

PSG.002 (-) control PSG.006
Vector (pSG.002) 3 uL 6 uL
Insert (pSG.006) - 0.5 uL
H2O 5.5 uL 2 uL
Ligase buffer 1 uL 1 uL
T4 ligase 0.5 uL 0.5 uL

Incubate in 20°C (microscope room) for 1h