cloning trouble

yangyigood at hotmail.com yangyigood at hotmail.com
Tue Nov 19 21:14:10 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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