Nested deletions with double stranded plasmids

Leonard Bloksberg preissj at clvax1.msu.edu
Thu Sep 16 12:21:00 EST 1993


In Article <1993Sep14.095248.29352 at infodev.cam.ac.uk> "arosenth at crc.ac.uk (Dr. A. Rosenthal)" says:
> 
> 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.  On
> 
> 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.
> 
First of all, please use the cariage return for posting messages.  It makes
them much easier to read.
	Now, about nested deletions, I have had lots of success with this
using the protocol from a kit from Boerenger Mannaheim.  Someone once bought
the kit and recorded the  protocol and ingredients.  We then just made up
the reagents ourselves and performed it many times for much less money.  It
has many advantages of speed and convenience because they have optimized 
each reaction to take place in the same buffer as the previous step, with
just the addition of an aliquot of buffer.  Hence, no phenol extractions
and precipitations in mid protocol.  This may not sound like much, but when
you have 20 to 50 timed reactions, it sure is convenient to have it as a 
1 tube reaction for each.  It probably is worth the investment the first
time.  Also, having done sequencing by the "multiple custom primer" method
also, I can say that exo deletions are just as easy to work with (unless you
are sequencing site directed mutants of the same clone over and over again).
Good Luck.
.
.			Dr. Leonard N. Bloksberg
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.
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