cloning cDNA libraries - (May/15/2007 )
I want to clone full length libraries in to gwiz empty from gene lantis so
So I prepare my cdna then digestion with not1 after that I did phenol purification then I ligat to my vector 1:1 ratio the problem when I transform I did not got any colony from the patrei dish I don't know what happen is there any one have an idea
It seems that your recombination efficiency is low. Assuming that your full length cDNA is in good quality, number of molecules per given mass should be much less than regular cDNAs that typically have ~50% or less of full-length molecules. In other words, if 1:1 ratio of ligates and vectors works for your normal insert, this condition may not necessarily optimal in this case. I would try 0.5:1, 1:1, 2:1 or even higher: this will give you idea of which direction you should go to get an optimal ligation condition. Hope this would help
thanx for your help but my Q Different of that
How's your positive plate?
well... it happened to me not too long ago, I got my new plasmid and did all the ligation and transformation step, but accidentally used the wrong plate with the wrong antibiotic. Got nothing, and so was my positive plate. In the end, I noticed that the resistance gene on the plasmid was kanamycin instead of what I presumed, ampicillin.
i'm sure i but the right resistance kanamycin