Methods Digest, Vol 41, Issue 2

[Virash Gupta]

I understand that you must have tried different combinations and have come
to depressing conclusions. I have passed through similar phase once. If DNA
template is showing good band on agarose, try following simple change but
using any RAPD primer.
- for 25 microlitre reaction use 5 microlitre of 1 mM  (or 1microlitre of
5mM) dNTPmix and amplify for ~ 30 reactions with annealing temperature of ~
38 degree C. If amplification is good multiband, your everything is good
except for primers (specific / random). The most common factor for non
amplification is use of higher conc of dNTPs. Try and tell.
On Fri, Oct 3, 2008 at 10:33 PM, <methods-request from oat.bio.indiana.edu>wrote:

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>   1. Re: PCR help please (Duncan Clark)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Fri, 3 Oct 2008 13:23:18 +0100
> From: Duncan Clark <blackhole from abuse.plus.com>
> Subject: Re: PCR help please
> To: methods from net.bio.net
> Message-ID: <fWzqcKB26g5IFAhu from abuse.plus.com>
> Content-Type: text/plain;charset=us-ascii;format=flowed
>
> Historians believe that in newspost
> <mailman.207.1222814603.29717.methods from net.bio.net> on Tue, 30 Sep 2008,
> Becky Pickin <vpireiner from gmail.com> penned the following literary
> masterpiece:
> > I have tested every
> >piece I can think of and have run out of ideas...none of my PCR reactions
> >have resulted in a band - specific or not.
>
> OK.
>
> Start with something simpler.
>
> Prepare a 2x PCR Master Mix minus primers and template i.e. Buffer, Mg,
> dNTPs, Enzyme and water.
>
> Now find someone with M13 forward and Reverse primers and a.n. pUC based
> plasmid. Could be pUC18, 19, a TA vector etc. with no insert.
>
> Using 25pM of each primer and 10ng of plasmid run 20 cycles of PCR using
>
> 94C 3mins
>
> 94C 5 secs
> 55C 5 secs      20cycles
> 72C 15secs
>
> 72C 7mins
> 13C hold
>
> Run 5ul on agarose gel and verify it has amplified. That proves your
> master mix is working. You should be able to dilute the plasmid a few
> 10fold dilns and still pick up a band noting that supercoiled ccc
> plasmid will be more refractory to amplification that linear plasmid.
>
> Assuming that works go to human DNA and pick a reliable gene to amplify.
> The following primers amplify fine - pinched from a Fermentas hotstart
> patent app.
>
> GAT GGG CTC TGA GAC TAT AAA GCC
> GTA GAG AGC TTC CAC CAG GTG TG
> 402bp product.
>
> Works fine with 55C annealing for 10secs and 72C for 30secs extension.
>
> Run 30ng, 3ng and 0.3ng for 35cycles loading 5ul of a 50ul PCR on an
> agarose gel. You should see a feint band at the 0.3ng (100copy) level
> and obviously much brighter bands at the higher levels.
>
> If your master mix is working and those primers do not work then I would
> blame your template. You can buy inexpensive human DNA as a test control
> from Sigma - D7011
>
> Duncan
> --
> I love deadlines. I especially like the whooshing noise they make as
> they go flying by.
>
> Duncan Clark
> GeneSys Ltd.
>
>
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> End of Methods Digest, Vol 41, Issue 2
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>



-- 
Dr V K Gupta
Sr Microbiologist (Molecular Biology)
Insect Molecular Biology Lab
Department of Entomology
Punjab Agricultural University
Ludhiana (Pb)-141004- India
M: 09815963210