cloning trouble

yangyigood at hotmail.com yangyigood at hotmail.com
Tue Nov 19 17:58:18 EST 2002


I am cloning a 5kb fragment into pBCKS vector which is chloramphenical resistant, 3.4kb.
The 5kb fragment coming from kpnI digestion of an original plasmid. I recover the fragment from gel. I cut the vector with kpnI followed by CIP treatment. Then use 9ul  5kb fragment, 1ul vector 3ul, T4 ligase buffer and 1ul T4 ligase and 1ulH2O to make the final volum to be 15ul. Incubation overnight at room temperature. then take 1ul from the ligation mixture to transform DH5 E.coli by electroporation. I use blue-white selection. I can see some white colonies in the original plates, and streak them in the selection medium to make the 2nd generation. then pick up white ones for DNA prep, meanwhile streak the 2nd white colonies in a fresh selection plate to make the 3rd generation. but the thing is the 2nd generation white colonies will become blue. and from the DNA miniprep I can only see empty vector. 
The thing interesting is i have successfully cloned the 1.2kb fragment into the 3.4kb vector. The 1.2kb fragment came from the digestion with kpnI as did for the 5kb guy.
The trouble is why I can not get the 5kb cloned into the vector?
with thanks



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