RAPD PCR breakdown

Deanne Bell dbell at fresno.ars.usda.gov
Tue Mar 23 16:58:29 EST 2004


Hey DrAnnie

Are you using some kind of 'hot start' type of Taq? Because whole 'lot
numbers' of these have gone bad in our lab.  I know you said that you
have tried 'fresh' Taq - did you purchase a new bottle or did you use a
different bottle that has been stored in the freezer awhile? 

Also do you have another template / primer system that you can run to
check whether it is your machine?  For example, if you are trying to do
RAPDs on monkey DNA, do you have DNA from a different species (say corn)
and SSR primers that are working?

Sometimes primers can get degraded when stored in plain water - but this
would not be the problem with freshly diluted primers.

If your template DNA has been sitting in TE buffer in the frig for a
long time, you might try heating to 65C 5-10 minutes incase of adhering
to utubes walls.  But you say you also tried 'fresh' DNAs.

Sometimes new companies change buffer constituents . . . etc.  Results
differ when you change brands of Taq also.  Maybe double check for too
much MgCl2.

I have also had my PCR machine blow a gasket.  You might check with tech
support to see if there is a way to monitor your PCR temperatures.

Hope this sparks some ideas for you to try
Dee Bell



-----Original Message-----
From: owner-methods at hgmp.mrc.ac.uk [mailto:owner-methods at hgmp.mrc.ac.uk]
On Behalf Of DrAnnie
Sent: Tuesday, March 23, 2004 10:07 AM
To: methods at hgmp.mrc.ac.uk
Subject: Re: RAPD PCR breakdown

Some info that might help:

1)  All of my primers are from Operon and are 10 mers.

2)  Some are older than others but even the fresh ones aren't working.

3)  I have over 100 primers, so I'm not going to list the sequences.

4)  I've used the same MJ Research PTC-200 this whole time. 

5)  After an intial 4 cycles that include a ramp down to a 37 degree
anneal, then there are typical 35 cycles with 94 degree melt, 37
degree anneal, and 72 degree extend with an additional 10 minute
extend at the end after all of the cycles are complete.

6)  Used the same 96 well plates and seals.  Tried to use the same
reagents, but companies have been sold and bought since I started
(including Operon) and so the reagents come with a different name, but
I am assuming the contents are the same as listed.

7)  Have tried fresh dNTP's, Taq, templates, etc. to no avail.

8)  Am about to try a run with snap freezing the primers.

Is there anything else I could tell you that would help?
---



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