ligation problems - cloning a PCR fragment
Posted 08 May 2001 - 09:00 PM
Thanks in advance
Posted 09 May 2001 - 09:00 PM
You dont mention anything about the PCR fragment except that it is 300 b.p. I assume that the restriction sites that you are cloning the fragment into are also in the primer sequences, when you amplified the original PCR fragment. I also assume that you cut the PCR fragment with these enzymes, Bst98I and BamHI first, then quiagen purified the DNA before cloning. How far into the primer sequences are these sites. If the BamHI is near the end of the primer sequence, at the 5' end, it probabley wont matter too much. But the other enzyme I dont know. If the Bst98I site in your primer sequence is too close to the 5' end, the enzyme may not be cutting all that efficiently. In which case, self ligate the PCR on its own, cut back with both enzymes (double digest?), purify DNA then reclone into your vector.
Of course, if the PCR fragment doesn't have these sites then your blunt end cloning the DNA into DNA vector with incompatible overhangs ! !
Posted 22 May 2001 - 09:00 PM
Posted 24 May 2001 - 09:00 PM
Posted 19 October 2004 - 02:09 PM