part27

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potata1
potata1's picture
part27

 I have a problem with cloning a cassette in pART27vector. I cut a cassette and open pART27 with NotI , link with t4 DNA ligase at 4 celsius for overnight, and transform the  E.coli DH10b,but  do not get the the recombinant. i don,t know why....

Biju
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 I have not worked on the

 I have not worked on the vector you mentioned.But I will check to find the restriction enzyme specificity(star activity, methylation); need of a quality of the ligase enzyme, suitability of the cell line for selecting the transformant  and method of selection of the recombinant.You need to eliminate each of the above as a robable reason by making a stad protocol and then changing only one parameter at a time. Ultimately, success of any cloning depends on the specific conditions of the lab and hence only you could design the best way to figure out a solution to the issue.
Hope this helps to soem extent.
Good luck with your expts.
Biju Joseph

potata1
potata1's picture
 thanks for your advices

 thanks for your advices