strange cloning result

Hiranya S. Roychowdhury hroychow at nmsu.edu
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
>was 
>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
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu

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