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OriT Knock Out

oriT Knock Out

  • By Xu Anting
  • Successfully ligated up- and downstream fragments of 600 bp to 1200 bp using overlapped PCR, but agarose gel also showed that there are unspecific bands (750 bp) in PCR products. Therefore I chose gel separation to get purified products.
  • Use BamHI and SalI to digest the fragment in 37 centigrade, overnight.
  • Ready to ligate fragments to pUC18 and have them transformed and sequenced.

Conjugation Test

  • By Liu Ting
  • pSC101 competent cells are ready. I transformed pUC18 to test their efficiency and will get the result tomorrow.
  • failed to purify the pSC101 plasmid (~9 kb) from E. coli. Will try again tomorrow.

Key & Lock by Yu Tao and Zheng Qinsi


  • Ligation transformants do not grow.
  • Re-ligation tonight, and overnight, plan to ligate under 16℃ tomorrow morning.
  • In case it is the XbaI/PstI digestion of the PCR product ends that failed. We will rePCR tonight, see below.

Transformation result of R0040<-J23078

  • There are no colonies in the experimental plate and the negative control plate but thousands of colonies in the positive control plate.
  • I think it is because of the inefficiency of the double digestion of J23078, though I can not rule other possibilities out.
  • I suggest do the double digestion, purification, ligation and transformation once more.

Double Digesting R0010 and J23066

  • Digesting R0010 with SpeI/PstI and J23066 with PstI/XbaI.
  • Digestion system contains:
0.5 µl     EcoRI
0.5 µl     PstI
10 µl      Plasmid
2 µl       10*H
8 µl       ddH20
20 µl      Total
  • Treat each plasmid with two 20uL systems(So totally 40uL system per plasmid).
  • 37℃ culutre for 8 hours.

R0010, J23066 Digestion Product purification

  • use Transgen EasyPure PCR Purification Kit / Quick Gel Extraction Kit.
  • 30uL after purflication, respectively

electrophorsis result

  • from left to right:
  1. Purified R0010 (vector) @ SpeI/PstI
  2. Purified J23066 (fragment) @ PstI/XbaI
  3. Precise Quantified Marker I

File:Peking 2007-7-25-r0010 and j23066 after purification.jpg

Ligation: R0010<-J23066

  • Ligate the J23066 fragment and $$$$$ vector
  • Ligation system contains:
7 µl       J23066 fragment
1 µl       R0010 vector
1 µl       T4-Ligase
1 µl       10 X ligation buffer
0 µl       ddH20
10 µl      Total
  • The negative control group contains no fragment but 7uL ddH2O instead.
  • 4℃ overnight.

PCR J23078

  • J23078 forward and reverse primer: stored as 50uM.
  • PCR system contains:
0.5 µL    Primer pf1
0.5 µL    Primer pr1
4 µL      dNTP
0.5 µL    Taq
5µL       10 X buffer
39.5µL    dH20
50 µl      Total
  • Primer final concentration 0.5uM.
  • Add a drop of liquid paraffin to each system.
  • PCR program setting:
Step1     94℃ 5min
Step2     94℃ 30s 
Step3     60℃ 30s
Step4     72℃ 30s 
Step5     Go to step 2 for 4 times 
Step6     72℃ 10min

J23078 PCR product purification

  • Use Transgen EasyPure PCR Purification Kit.
  • 50uL after purflication.