PCR templates on Automated DNA sequencers

Shaun Tyler styler at HPB.HWC.CA
Sun Feb 25 16:22:10 EST 1996


We sequence a large number of PCR products and use the same method as y=
ou (i.e. gel purification=20
of the template and dye terminator sequencing).  We have not really eva=
luated alternative=20
methods, such as asymmetric PCR, but they seem like a lot of extra work=
 which is probably not=20
necessary.  As I=92m sure you know, with fluorescent sequencing, the qu=
ality of the template is=20
extremely important and these reactions can be quite finicky at times. =
 Although the same method=20
can be used to prepare different templates the quality of the templates=
 is not always the same (I=20
personally believe the difference is a result of the combination of bar=
ometric pressure, lunar=20
cycle and astrological alignment of the stars).  Whatever the cause, wh=
en a particular PCR=20
template or any other template sequences poorly,  we generally just rep=
eat the sequencing=20
reaction and try again (the stars were probably out of alignment).  If =
the sequence is still=20
lousy try making a new template and start from scratch.  We often have =
people submitting=20
templates which are just garbage but when we prepare them they sequence=
 just fine.  I have=20
watched these people prepare the templates and I can=92t see anything t=
hey are doing wrong.  Some=20
people just seem to have the =93gift=94 for doing this stuff.  Another =
consideration is the primer=20
used for sequencing.  We often see templates sequence well with one pri=
mer and not with another.

I no longer do the routine sequencing so I=92m a little out of touch wi=
th some of the quirks that=20
come up but if you like I can put you in touch with our tech who does o=
ur sequencing to discuss=20
the matter further.

Shaun Tyler
DNA Core Facility
Laboratory Centre for Disease Control
Health Canada

Ph#:  (613) 941-6441
FAX#: (613) 957-1358

E-mail:  styler at hpb.hwc.ca

PS.  As a new ABI user you may not be aware of this but ABI runs =93Use=
r Meeting=94 about twice a=20
year.  We have found these to be very helpful in discussing our own pro=
blems with others in the=20
field and to hear about some of the problems (and solutions) other user=
s have run into.

PPS.  If you continue to have problems try sacrificing barnyard animals=
 to the Gods.  Start with=20
the small ones and work your way up.

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