i really need your help since i've been having this problem for a few months...i have amplify lac Z gene (3 kb) using E. coli that have that properties and i manage to get the fragment. i add RE site which is Sal 1 on both sites ..and i ligate them overnight using amersham T4 DNA ligase. with ratio 1:3, 1;1, 3;1, 1;7, 7:1, 1:10, into a plasmid size (3.3 kb) which have kanamycin resistant gene, and tranform them. The transformation is good because i got a lots of colonies on positive control. the problem is that all the colonies from my clone that grow are white in colour while i desperately need the blue ones..please help me..
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cloning of lacZ
1 reply to this topic
Posted 24 April 2003 - 08:25 AM
I have been trying to clone a Sal I digest. It looks like Sal I is giving some sort of star activity. I am unable to clone a 450 bp fragment in 10.5 kb vector. I dephosphorylate the vector. Therefore, I don't get any colonies at all. But your problem may be solved by dephosphorylation by CIP or shrimp alkaline phosphatase. You wouldn't see any self ligation in that case.