A new band after CIPing

Antonin Tutter atutter at aim.salk.edu
Sun Oct 4 00:18:19 EST 1998


>Hi,
>I cut my vector (pIVET8) with BglII, gel purify it (Nusieve Agarose), CIP
>it. To deactivate the CIP, I used Proteinake K an d SDS and incubated at
>55 C for 30min. Phenol-chloroforme extraction then precipitation with NaAC
>and ethanol.
>
>My surprise is the apparition of a new band with a high molecular weight
>(>30KB) while my plasmid is around 10 Kb. I don't know what happened. did
>my digested plasmid polymerize, I really have no clue.
>
>So I was wondering if anyone had that sort of weird size enhancement or
>not? and is there any explanation to that phenomenon?
>

Why don't you simply heat kill the BglII, CIP treat and THEN gel purify?  As
for the high mw band, perhaps one of your reagents is contaminated with DNA.



_______________________________________
Antonin Tutter
Salk Institute for Biological Studies
RBIO-J
10010 N. Torrey Pines Rd.
La Jolla, CA  92037
email:  atutter at aim.salk.edu
web:  http://www-biology.ucsd.edu/~atutter/




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