strange cloning result

Hiranya S. Roychowdhury hroychow at
Wed Mar 29 12:22:58 EST 2000

At 11:00 AM 3/29/00 -0600, Chris LaRosa wrote:
>> Anyway, that is why I am such a die-hard advocate of v~OH. It eliminates a
>What do you mean by v~oH???

dephosphorylated vector ;>

>Also someone perhaps in this thread mentioned problems with ciap.....I
>wondering that since I am using CIAP, and have been having problems
>cloning into CIAP treated vector,  what are the pitfalls of CIAP...?

Yes, CIAP (aka CIP) can cause problems during the ligation rxn if present.
Usually, the following method works really well:

After CIP treatment, add EDTA to 10mM and SDS to 0.01%.
Heat the tube at 65 C (20min) or 75 C (10min).
Extract 1x w/ P:C:I
Extract  1x w/ C:I
re-ppt the digested and dephosphorylated vector. 15min on ice, spin 20min...
resuspend the vector in minimum TE and run out on a gel
purify from the gel using your favorite method.

That is why I have switched to SAP... heh!heh!
However, I still gel-purify my vector preps. IMO, vector prep is the key to
obtaining good ligation/transformation. 

Dr. Hiranya Sankar Roychowdhury
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at


More information about the Methods mailing list