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Problem with cloning by dephospharylation - Problem in cloning (Jan/22/2005 )

HI, I am a newcomer in molecular cloning and met some problem in my first project. I hope you could give me some advise.
I digested a plasmid called pUASt(9kb) with NotI(single cut site), dephospharylated it by CIAP(Takara), run it in low-melting agarose and recover it by kit(promega). The insert is 2.5kb which is also digested by NotI. I ligate them using ligase(takara) at 16C, overnight in 25ul. The plasmid is about 20-50ng and insert is about the same weight or more. I did transformation using JM109(takara) whose effeciency is about 10e8.
I always got no colony. Here is the controls:
Plasmid digestion: OK
Other clonings using two different endonucleases:OK
Plasmid self ligation after CIAP:0 colony
Plasmid self ligation without CIAP: around 1000 colonies
Plasmid without CIAP but with insert: arount 100 colonies
Plasmid dephospharylated by 10X diluted CIAP: 0 colony

Can you tell me what further modification should I do, or other controls should I do to find out the problems?
Thank you very much


i'm no expert but your controls look godd to me...did you try different ratios of insert to vector? and also - is it possible your insert encodes something which is fatal to the cells?


Dear leahf, the insert is not expressed in the E. Coli. I have tried the ratio from 1:3 to 1:10. If I want to make some modifications, should I increase the amount of vector or decrease it? Does the volume of the ligation system matter?


Hi mammoth - i wish i could help but i've no more ideas! i don't know about the volume and amount vector - maybe post a new question about it?
Be strong & don't give up!


Try to remove CIAP with Phenol:chloroform before the ligation. CIAP can to inhibit the ligation reaction.
Too try cut the plasmid an ligate this to your insert without CIAP treatment, but with a molar ratio 1:2 or up (plasmid:insert).

PD.- sorry for my poor english