{Q} Effective Inverse PCR?

David Micklem dmicklem at cmgm.nospam.invalid
Wed Aug 2 18:14:46 EST 2000


In article <8m8usc$hs2$1 at nnrp1.deja.com>, Andreas Firzinger
<a_firzinger at my-deja.com> wrote:

>I just found two papers in recent BioTechniques (Vol.28, No.5 (2000)).
>Never did inverse PCR before, but I'd have to during next three
>month. However, what really surprised me was the HUGE amount of
>T4 ligase the authors used: 75µl volume with low DNA-concentration
>to achieve a high rate of self-ligation (2.5ng/µl) and 4units
>T4 ligase/µl, this means 300 units T4 ligase in one reaction!!
>
>Ciao,
>
>Andreas

The Drosophila genome project protocol at

<http://www.fruitfly.org/about/methods/inverse.pcr.html>

only uses 2 weiss units/reaction.

Their protocol has worked well for me, for doing inverse PCR off
transposons. I can't see why it should work any differently for inverse
PCR of any other sequence in the genome.

I suppose that for organisms with a much larger genome everything might
need to be scaled up a bit.

David

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D.R. Micklem,          Time flies like an arrow... Fruit flies like a banana. 
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