Fw: sequence services

David Micklem 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:

<snip>

>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
spinning it.

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.

HTH,

David

-- 
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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