Automated sequencing problems (ABI)

Harry Witchel Harry.Witchel at
Tue Dec 16 08:13:19 EST 1997

Hi folks!

I currently use an in-house facility for sequencing (ABI 377, cycle
sequencing), which is quite reasonably priced.  However, it is extremely
common for them to be unable to sequence my templates.  This is often
true for standard primers (eg M13 uni and reverse) as well as other
primers.  My templates are often quite GC rich (65%).  I purify my
plasmids using Promega’s Wizard Plus kits, which have (at times) given
me very good sequence data with the same sequencing facility.  I always
use the same strain of bacteria (XL1 MRF’), although every few months I
have to make a new batch of competent bacteria.

The inconsistency of my results disturbs me, and I want to increase my
quality control.  I always check my plasmids before sending them out for
sequencing (thus checking for plasmid concentration & for the ability to
be restriction cut).  I am now trying to keep track of all the variables
that might affect sequencing, which so far include: Date, Miniprep type
and batch #, Sequencer, Primer, Bugs, Medium, Vector.  If anyone knows
of any other important variables, I would appreciate it.

Finally, as I am trying to do some quality control and cost savings, I
am trying to figure out what is worth doing.  I have used Qiagen
Tip-20s, and they cost 6x as much as the Wizard preps, and at times they
did not work (but I never did a formal analysis of why or what percent
failed compared to Promega).  Any help managing this problem and
increasing my sequencing success is appreciated.

Harry J. Witchel, Ph.D.
Dept. Physiology
Medical School
Bristol  BS8 1TD

Harry.Witchel at

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