I am in big trouble with ligation. I purified DNA from agarose gel with QIAquick Gel Extraction kit, I processed a tailing DNA fragment and then ligation with pGEM-T easy vector. I got blue colonies at all. I tried many times but I failed. Do you have any suggestion to improve my problem?
check the blue colonies whteher they contain a cloned fragment. Sometimes the colonies are light blue and may contain a cloned fragment because they are in frame with the Lacz gene
Hi Tuyet....
The pGEM-T easy vector is a T-tailed vector...where 3'-end T has been chemically added to the blunt end digested vector. This additional 'T' prevents the self-ligation of the vector and as a result there should not be any (or very few) colonies containing self ligated plasmids (or blue clones in this case)....This indicates that your vector is getting self ligated....Such things are observed with T-vectors if the vector is very old and has undergone repeated freeze-thaw cycles....and over the time some-how the T-overhangs have been cleaved.....
Since you have seen this problem.....I will suggest you to first do just a 'self-ligation' ...of the vector...if you are getting a large number of blue colonies...probably your pGEM-T easy vector has gone bad...get a new one in this case.....so don't do several controls....just do a self ligation first...and if the vector is fine.....'you don't even need blue/white selection..
bye
Hi I am not very sure but I did ligation with takarra ligation kit d6020a and it works wonders I hope it will work as before I was facing the problem of not getting any positive colonies too....good luck
check the blue colonies whteher they contain a cloned fragment. Sometimes the colonies are light blue and may contain a cloned fragment because they are in frame with the Lacz gene
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