PCR help please

[Duncan Clark]

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.