ABI sequencing primer question

[Eric C. Anderson]

howdy,

i run a small DNA sequencing facility here using the ABI 373 autosequencer
and Taq Dye-Terminator chemistry for the reactions. i recently ran across
a problem with one customer who is trying to use cloning primers with
restriction sites built into them (NotI and SpeI) to sequence her clones.
the NotI containing primer is 32bp long with only the 3' 20bp actually
hybridizing to the plasmid, and the SpeI primer has only 22 of 32bp which
hybridize.

her sequencing isn't working at all and now that i know this information
about the primers i'm wondering if this might be due to the fact that only
2/3 of the primers are hybridizing to the plasmid. anybody have any
experience or information with this type of situation? any help would be
greatly appreciated.

thanks,

eric

--
"i don't know what caffeine does for you, but i'm pretty sure that without it your head caves in."

eric c. anderson ande...@pharmdec.wustl.edu
dept. of molecular bio. and pharm. (314)362-3963 (lab)
washington univ. school of medicine (314)362-7058 (FAX)
660 s. euclid box 8103
st. louis, mo 63110

for a comprehensive list of bike related web pages... http://pharmdec.wustl.edu/~anderson/anderson.html

[Peter Myler]

Eric:

We have had similar experiences here. Primers with a lot of extra 5'
sequence generally do not make the best sequencing primers. I would
suggest making new sequencing primers (they are very inexpensive,
nowadays), if more than 5-6 bp don't match at the 5' end.

Peter

===============================================================================
Peter J. Myler phone: (206) 284-8846x332
Seattle Biomedical Research Institute FAX: (206) 284-0313
4 Nickerson Street e-mail: MYL...@U.WASHINGTON.EDU
Seattle, WA 98109-1651
===============================================================================

[Eric C. Anderson]

In article
<Pine.A32.3.91j.95062...@homer25.u.washington.edu>,

Peter Myler <myl...@u.washington.edu> wrote:
> We have had similar experiences here. Primers with a lot of extra 5'
> sequence generally do not make the best sequencing primers. I would
> suggest making new sequencing primers (they are very inexpensive,
> nowadays), if more than 5-6 bp don't match at the 5' end.

peter,

thanks for the help. something that i decided to try was to increase the
concentration of primers to make up for the fact that only 2/3 of the
primer was actually binding to the template. it worked on the shorter of
the two primers but not the longer one. unforturnately i will have a
tough time talking this lab into buying new primers, regardless of cost.
they actually wanted to try sequencing this template with a 50-mer primer
that had a 15bp overhang. oh well.

[Peter Myler]

Eric:

It almost costs more to do a single sequencing reaction ($15-25, if
done internally, >$50 if external) than it does to buy a short sequencing
oligo at
the 0.02uM scale (~$30 for a 20-mer) and certainly less than the cost of
having to spend all the time dealing with crappy sequence.