IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-31
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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.
- PCR program condition 1: 94℃ 5min, 94℃ 30s, 51℃ 30s, 72℃ 30s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
- 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:
- 37℃ @ program 1
- 30℃ @ program 1
- 37℃ @ program 2
- 37℃ @ program 2
- marker
OriT PCR product purification
- use Transgen kit.
- 40uL after purflication
electrophorsis result
- from left to right:
- 37℃ @ program 1 before purflication
- 30℃ @ program 2 before purflication
- 30℃ @ program 1 after purflication
- 37℃ @ program 2 after purflication
- marker
- the left two channels are used to re-test the aboabnormal line that showned on PCR product electrophorsis test.
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.