Summary: Automated sequencing solutions (ABI)
Harry.Witchel at Bristol.ac.Uk
Thu Dec 18 10:13:58 EST 1997
"The compendium of useful advice received concerning making minipreps for
ABI automated sequencing of GC rich templates."
First I would like to thank all the generous and experienced people who sent
recommendations and wisdom including: Bill Buikema, Caroline Szymeczek-Seay,
Todd Martinsky, Ed Castro, Greg Hawkins, and Zal Suldan. What follows is a
brief compendium of the useful and sometimes disparate ideas presented.
Most trouble with automated sequencing is due to one of three things
the sequencing reaction,
the primer, or
the template quality.
TEMPLATE QUALITY. Many commercially available kits exist, the two most
popular being Promega Wizard Plus kits and Qiagen Tip-20s. (Other kits
mentioned included Omega Biotek, Qiagen Spin preps and the ArrayIt PCR
Purification Kit). The Promega kit is 1/3 the price of the Tip-20s, and it
is also significantly faster; however, quite a few people reported
difficulties or inconsistency using the Promega kit, while everyone using
the Qiagen kit was satisfied 100% of the time. I wondered if this is a case
of you get what you pay for?
However, Bill Buikema, who is intimately involved with a sequencing
facility, suggested that brand names were probably less important than what
you do to the DNA. Two typical problems for miniprepped DNA are:
Too much salt
Co-purification of bacterial polysaccharaides
Bill suggested two fixes for these problems. To get rid of the salt, he
recommends 1-2 isopropanol precipitations followed by an ethanol wash at the
end of the protocol. (using 0.5 vol of 7.5 M NH4Oac and 0.6 vol of
isopropanol do not chill the precipitation). Not surpisingly, the Qiagen
protocol does include an isopropanol precipitation, whereas the Promega
protocol does not. I personally will dilute my 50 ul of Promega eluate with
another 350 ul of TE before precipitating.
To get rid of bacterial polysaccharides, Bill recommends an initial wash of
the bacterial pellet with .5 M NaCl at the very beginning of the protocol
(which is simple and does not require any tube changes). The polysaccharide
problem may explain why some bacterial strains are easier to sequence than
others (the easy strain mentioned was DH5 alpha).
GC RICH TEMPLATES: One person recommended either 5% or 10 % DMSO in the
sequencing PCR reaction as a requirement to get longer reads. A technical
director at Epicentre recommended their kit for GC rich sequencing
(Epicentre Technologies sells a sequencing kit called Sequitherm Excel II
that is designed to sequence through high GC templates).
PRIMERS: Only one person commented: We started using the universal and
reverse primers but found them less than optimal for 2 reasons: One, the
primer was about 100 nt from the insert and this resulted in wasted
sequence. So, I designed a primer that was about 20 nt from the insert.
Two, the melting temp of the primers was too low and we were forced to run
the annealing step at a 45 (or so) temp. With the new primers I was able to
run the reaction at 55 or 60 which resulted in cleaner runs with more info.
SEQUENCING REACTION: While it is definitely true that the quality and
consistency of ones sequencing reaction does influence the length of the
sequence read (but not whether the sequence starts), the only recommendation
was to avoid oil carryover.
Many thanks to one and all again,
Harry J. Witchel, Ph.D.
Bristol BS8 1TD
Harry.Witchel at Bristol.ac.uk
More information about the Methods