cloning trouble

yangyigood at hotmail.com yangyigood at hotmail.com
Tue Nov 19 18:41:51 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 tThe 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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