cloning NdeI cut products

Richard P. Grant spam.blocked at delete.this.cmtech.co.uk
Thu Feb 12 03:14:41 EST 1998


John, 

OK, so how is your uncut plasmid DNA prepped?   A lot of methods give DNA that
won't cut with NdeI (allegedly) but it looks like yours is cutting.
Sounds like you're getting nibbling of the ends of the cut plasmid.  You could
try adding spermidine to 2mM and BSA to 150 ug/ml in your enzyme digest.

Alternatively, the gel purification method could be screwing up your ends, it
has been known.  Yes, we sell a kit for doing this too (-: , but have you
actually tried the religation without gel purification?  You might get high
background but at least you'll be narrowing down the possibilities.


btw, which vector is it?

R

PS Substitute "you" for "the cyber-illerate one" :-))

> 
> my benchmate (the cyber-illerate one 8^) is trying to do a XbaI-NdeI
> cloning. After much failure, he tried single cutting the vector with XbaI
> and NdeI, and then re-ligating. The vectors cut, as confirmed by gel
> isolation of the band. However, after passing the gel slice over a qiagen
> gel-purification column (I know, but it has worked in the past), ligation
> of the XbaI-cut vector gave transformants, but ligation of the NdeI-cut
> vector did not. Same amounts, same cells, same vector, same everything
> except the res'n enzyme.
> 
.

-- 
Richard P. Grant MA DPhil  | Cambridge Molecular Technologies
Senior R&D Scientist       | rgrant at cmtech co uk
Tel: +44 1223 508345       | http://www.cmtech.co.uk/



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