IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-25

=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=

R0040<-crRNA

 * 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

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 End

J23078 PCR product purification

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