PCR cloning in phage
tyarovin at blue.weeg.uiowa.edu
Thu Jan 22 15:04:04 EST 1998
I wish to clone PCR products in landa ZAP II. I used non phosphorylated
primer for the PCR, so I assume that 5' ends of my DNA is composed by
primers mostly and non phosphorylated. I polish my DNA with T4 DNA
polymerase to cut 3' overhangs generated by Taq and ligate
hemiphosphorylated (at blunt end) adaptors. If I am not wrong ligase can
ligate only one strand of my DNA to adaptors in my case and leaves a nick
on the other strand. Eventually I phosphorylate sticky ends of
adaptors-DNA and ligate to vector. May the nick be a reason to fail in the
cloning because of whatsoever reason? I know there are a lot of kits that
allow to do transformation with that nick, but what about transfection?
Any comments encouraged.
Timur Yarovinsky, Ph.D., M.D.
Oakdale Research Park, B141 MTF
2501 Crosspark Road
Coralville IA 52241-8802
tel. (319) 335-4612
fax (319) 335-4347
e-mail: timur-yarovinsky at uiowa.edu
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