I need to digest a vector (single digestion, sticky ends) and dephosphorylate before ligation with the insert and then transformation into E.coli. The problem is I don't have much of the vector to begin with (v. low yield) and I keep losing alot of DNA in purification. Do I absolutely need to purify between digestion and dephosphorylation? And do I really need to purify again before ligation? I've heard it's not neccessary, but what about the mix of buffers you get? I'll heat inactivate the RE, but do I need to do this with the AP? Any suggestions would be appreciated.
2006-11-22
22:38:36
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2 answers
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asked by
cheetara_2001
2
in
Biology