cycle sequencing with 35S??

jmv at canctr.mc.duke.edu jmv at canctr.mc.duke.edu
Wed May 26 16:06:03 EST 1993


In article <C7AB34.35M at ccu.umanitoba.ca>, jrichmn at ccu.umanitoba.ca (Joy Richman) writes:
>Dear Netters,
>
>Has anyone out there got cycle sequencing to work using 35 S? 
> [......]
Yes! It works, using the direct incorporation protocol on plasmid DNA
(ie, use alpha-35S-dATP). We've done it using the Promega fmol
kit, which we use for most sequencing done here: follow given 
instructions (`1 ug plasmid). Amersham and several seq. kit makers
tell me that the end-labeled primer (kinased) protocol does not
work with gamma-35S-ATP.

When seq. very small or very large templates (PCR prods. or cosmids
or lambda DNA), incorporation rxs. often require unrealistically
concentrated template DNA (eg Sequenase=5-10 ug cosmid DNA in 7 ul
buffer, max); therefore, cycle sequencing, which we find requires only
300 ng cosmid, 500 ng lambda, 150 ng plasmid, or less than 1/3 of
PCR prod. excised from agarose gel. I too hate 32P, so I use 33P.
(gamma-32P-ATP from NEN or Amersham is 4x as expensive, but can be
used successfully when over a month old--use 2x as much, usu. 1 ul
per labelling rx.--labelled primer can do over 6 seq. rxs.) 33P also
yields bands as sharp as 35S & seqs. are much easier to read than those
from 32P rx.

Most major biotech companies now offer cycle sequencing kits, & from
comparing kit manuals & tech-reps'comments, are the same thing. I've tried
BRL, Promega and Perkin-Elmer-Cetus. Promega is the only kit that includes
kinase & buffer for primer end-labelling (it's not the best PNK: we usu.
use BRL or Pharmacia PNK anyway), for the same price. Stratagene, USBC,
NEB, and Amersham advertise similar kits & even the same suggested PCR
settings. (You have to play with these to optimize for your primers.)
The most user-friendly kit is from PEC: all reagents are diluted so that
you set your pipetman on 4 ul for all steps in the protocol.

*************************************************************************
OK, now that this FAQ has an A; could someone who has actual results with
the Promega silver-stain sequencing kit please post his experiences?
The cat ate some cheese & is waiting at the mousehole with baited breath.

(-:
DB
Duke U Med Ctr/Neurology



More information about the Methods mailing list