Nested deletions with double stranded plasmids

Dr. A. Rosenthal arosenth at crc.ac.uk
Tue Sep 14 04:52:48 EST 1993


Hi guys...this is my first time on the network for a long time, so I hope everybody has lots of good advice!

I have a 7 kb fragment I want to rapidly sequence and want to avoid either shot-guning or primer walking.  One solution appears to be make nested deletions enabling sequencing off a single universal primer.  I notice there are at least a couple of kits out on the market to do this (from Stratagene and Pharmacia); both usinge Exo III for the deleting part and S1 and mung bean nucleases respectively for blunt ending.

So....Does anyone out there have any experience with this technique (sucessful or otherwise!!).  What about the relative merits of S1 over the mung bean enzyme? How time consuming is the procedure?
Is it worth the $$? Anything to pay paricular attention to, eg how easy is it to control the exo to get 
a broad range of fragment sizes?


Any answers gratefully received

Cheers.



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