minipreps for sequencing

txpljfg at UABCVSR.CVSR.UAB.EDU txpljfg at UABCVSR.CVSR.UAB.EDU
Fri Mar 4 17:43:12 EST 1994


We just compared several methods of miniprep plasmid preparation in
order to determine which was the easiest and gave the best results for
sequencing.  It should be noted that we are interested primarily in
short sequences that are within 200bp of the primer.  We generally
don't need more than 50-60bp of readable sequence for our purposes. 
However, the sequence must be reasonably unambiguous because we are
interested in reading frame usage particularly in the gene segment
junctions of T cell receptor transcripts (a highly GC rich area)

We compared plasmid preps from 1) The boiling miniprep method from
maniatis using the standard sequenase denaturation protocol and also
by boiling the plasmid in NaOH in the presence of the primer for two
minutes; 2) RPM minipreps from Bio101; 3) Insta preps from 5' to 3';
and 4) a scaled down version of the PEG precipitation protocol from
Maniatis (all references to this text are for the newer edition).

All preps were done from the same cultures.  The clones consisted of
150-200bp inserts in pUC19 in XL-1 blue cells grown in Circleprep
medium (from Bio101) for approx 18 hours.

The PEG ppt protocol didn't work at all, so it is probable that we
botched it.  The rest of the miniprep methods all gave good sequence,
readable for at least 150bp.  There was no significant difference in
readablility between any of these methods.  My tech tells me that the
easiest method was the Insta Prep method, taking only a few minutes to
prepare three templates.


Our interpretation of these results is that if expense is a major
consideration, then the boiling miniprep method is perfectly suitable
for most routine short-range sequencing.  If your objective is to
sequence as many templates as possible in a short time, then the insta
prep works well.

Our largest problem so far has been stops at some of the GC rich areas.
 The usual remedies (formamide gels, deaza analogs) haven't been as
effective as we had hoped.  We intend using TdT to try and remedy this
problem.  Any other ideas would be greatly appreciated.

==============================================================================
James F. George, Ph.D.              "Back off man, I'm a scientist"
Department of Surgery                --Bill Murray
University of Alabama at Birmingham
205-934-4261 voice
txpljfg at uabcvsr.cvsr.uab.edu
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