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

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

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.

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.

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.