FW: RAPD PCR breakdown

Deanne Bell dbell at fresno.ars.usda.gov
Wed Mar 24 10:51:21 EST 2004


The dialogue continues . . .
TTFN
Dee Bell

> -----Original Message-----
> On Behalf Of DrAnnie
> Gibco is now sold by Invitrogen.  Can anybody suggest the best Taq for
> RAPD's?

I do not know that there is a "best" Taq - but certainly there are
differences between companies and different Taqs work better under
different conditions.  I have found that it is important to stay with
the same Taq all through an experiment.  If you change Taq, you need to
go through all the optimization steps again like optimizing
concentrations of  [mgCl] & [DNA] - maybe some adjustments to annealing
temperatures.


> 
> We have a 16S bacterial amplification that works beautifully on the
> same machine but both our RAPD amplifications for beetles and fish are
> not working.

Well, if you are using the same machine and the same reagents for the
bacterial amp, it seems that it boils down to problems in your template.


Another thought here is PCR inhibition.  I work with plants and insects
mostly and I have had a lot of problems with interfering substances in
the DNA preps.   One way to test for this is to do a [DNA] optimization
especially looking at decreasing the amount of template that you put
into the PCR mix.  It is kind of counter-intuitive because one would
think that 'if I am not getting amplification, I should increase the
amount of template'.  Actually, by decreasing the amount of DNA prep you
get down to a place where an inhibiting substance gets diluted out, but
there is still enough DNA to amplify.

> 
> We've just tried snap-freezing the primers, but I don't have those
> results yet.  We do dilute those in water.

Rookie Question: I was wondering if you could elaborate on this
'snap-freezing'?  I am not familiar with the term, so I am not sure what
this is.

> Any other suggestions are welcome!

Hope this helps
Peace
Dee
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