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

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
End

J23078 PCR product purification

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