Fw: sequence services
dmicklem at cmgm.nospam.invalid
Thu May 10 09:26:30 EST 2001
In article <200105092135.f49LZTB17794 at ns.qnis.net>, Deanne Bell
<dbell at qnis.net> wrote:
>BUT, let's say I have a DNA band from a PCR that specifically amplifies the
>large sub-unit ribosomal DNA. I can be pretty confidant that all the
>fragments in that band are the same. Then I could clean up DNA with a PCR
>clean up kit and send it to someone for sequencing along with an aliquote
>of my primer.
Yup! It doesn't even need to be _that_ clean. I run the PCR reaction
out on a gel in a big slot (about 1cm). Cut it out over long-wave UV
and pop it into a Millipore UltrafreeMC spin-filter (or make your own:
See Freeze/Squeeze type protocols discussed here prevously). Spin for
a couple of minutes and use for sequencing. I usually run my PCR
products out on a gel anyway, to find out if they've worked, so the
only extra work prior to sequencing is cutting out the band and
I find I get about 20-25ul of liquid after the spin. From a moderately
bright band this has been enough for me to get at least 3 sequencing
reactions using BigDyes. You can use the same primers you used for
PCR, but you might get _slightly_ better results using nested primers.
I didn't have any problems with using the PCR primers when I was doing
this. An advantage of sequencing direct from the PCR product is that
you can detect polymorphisms/mutations - I was PCRing up introns from
genomic DNA from fruitflies heterozygous for a mutation in my favourite
gene. Point mutations and small insertions/deletions were easy to spot
directly from the sequencing traces.
D.R. Micklem, Time flies like an arrow...
Dept. of Anatomy, Fruit flies like a banana.
Cambridge University, Email:dmicklem at cmgm.stanford.edu
Cambridge, UK Phone: +44 (1223) 333776
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