IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-31

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Contents

Tandem OriT By Qu Mingzhi

PCR OriT

  • get F-OriT primer, primer vf,vf2,vr,pf1,pr1 stored as ~100uM.
  • PCR system contains (totally 50uL):1uL Primer pf1, 1uL Primer pr1, 4uL dNTP, 0.5uL Taq, 1uL OriT template, 37.5uL dH20, 5uL 10X buffer,
  • use different PCR program to test efficiency.
  1. PCR program condition 1: 94℃ 5min, 94℃ 30s, 51℃ 30s, 72℃ 30s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
  2. PCR program condition 2: 94℃ 5min, 94℃ 30s, 55℃ 30s, 72℃ 30s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end .
  • Primer final concentration 1uM.

electrophorsis result

  • from left to right:
  1. 37℃ @ program 1
  2. 30℃ @ program 1
  3. 37℃ @ program 2
  4. 37℃ @ program 2
  5. marker

Image:Peking_2007-7-31_PCR_OriT_T51_37_30_T55_37_30(2).jpg‎

OriT PCR product purification

  • use Transgen kit.
  • 40uL after purflication

electrophorsis result

  • from left to right:
  1. 37℃ @ program 1 before purflication
  2. 30℃ @ program 2 before purflication
  3. 30℃ @ program 1 after purflication
  4. 37℃ @ program 2 after purflication
  5. marker
  • the left two channels are used to re-test the aboabnormal line that showned on PCR product electrophorsis test.

Image:Peking_2007-7-31_PCR_purflication_oriT.jpg‎

transformation OriT

  • Use pEASY-3 as cloning vector .(Amp+)
  • tramsformation system contains :3uL PCR product(after purflication), 1uL pEasy-T
  • the Culture Dish(LB/Amp+) are trated with 5uL 1M IPTG & 40uL 40mg/mL x-Gal.
  • NEXT DAY: 10 white cloney received.

Lock & Key By Yu Tao

oriT Knock Out

  • By Xu Anting

Competent Cell preparation

  • After activation of three strains (F/R/S) plates, shake them with LB liquid (pSC101 in Tc+) in 37℃ for 16 hours.
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