IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/08/16

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(Entry title)
(Ligation of pTet)
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==Ligation of pTet==
==Ligation of pTet==
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We ligated pTet (annealing product) with pSG.002 (E,S)
+
We ligated pTet (annealing product) with pSG.002 (E,S), Kan resistant
===Protocol===
===Protocol===

Revision as of 11:42, 17 August 2012

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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

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

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