DNA got degraded after klenow, CIP and Phenol extraction - (Apr/22/2005 )
Hi friends. I need to clone an insert(1.3kb) in to a vector (6.6kb). I am following this path: Restrict Digest insert and vector>Gel elute (electroelution or freeze-thaw)>Phenol:Chl extract>Klenow end filling(NEB klenow exo-)>CIP(NEB) for vector>Phenole:Chl extract>Blunt end ligation. Now my DNA got degraded after 2nd Phenol:CHl extraction(After klenow and CIP) WHat could b the reason? I had started with lot DNA(Bulk digestion, ~16ug in 50ul reaction. PLease suggest. Thank u.
i don't know about your method of gel exteraction. did you check dna quality after this?
For CIP, one hour is ok for achieving more than 95% of dephosphorylation. I don't know effects if too long step. But i don't know where can be your problem.
Try to get fresh buffers caus i suspect presence of DNase in one of it...
i hope that helps.