Nested deletions with double stranded plasmids

William R. Morgan wmorgan at ACS.WOOSTER.EDU
Tue Sep 14 09:17:28 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 ou
>
>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.

I follow exactly the protocol described in Henikoff,S. (1987) Methods in
Enzymology, Vol. 155, p. 156-165.  It works great; the only variable seems
to be the rate of ExoIII digestion which you can check by agarose gel
electrophoresis (as described in the paper).

FYI:  My fragments are routinely cloned into pBlueScript and then
transformed into JM101.

Good luck,
Bill Morgan

William R. Morgan
The College of Wooster
Department of Biology
931 College St.
Wooster, OH  44691
Phone:  216-263-2026
FAX:    216-263-2378
E-mail: wmorgan at acs.wooster.edu




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