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.
- 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
- Purified R0010 (vector) @ SpeI/PstI
- Purified J23066 (fragment) @ PstI/XbaI
- Precise Quantified Marker I
Image:Peking 2007-7-25-r0010 and j23066 after purification.jpg
- 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.
- 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
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.