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....
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.
thanks for your advices