PCR amplification problems could use some help

Robert Hartley r.hartley at freeuk.com
Mon Jan 3 20:16:32 EST 2000


In message <3870084A.80B56A6C at navix.net>
          Chris LaRosa <plarosa1 at navix.net> wrote:
I agggree with almost all things Chris has said.

I see you are trying a PE9700.
Are you working on Max mode? If so try 9600 mode to slow the thing down.

We have a couple of these and Robocyclers and half a dozen MJR PTC200.
I can be impartial since I use a Rapidcycler. :-)
For the PE I would make the ramp to 94 fast and the ramp to annealing fast
but slow the thing down between annealing and elongation. Ramp at 1 deg/sec.

I see you are using an Ab? THis is going to be insulting so I must appologise
in advance BUT; do you do a PRE-PCR to release the AB.

Try this

94-5/10mins
then 30 cycles
94-30 sec ramp down at max
55-30sec ramp up at 1deg
72-1.5min ramp up fast
then 72 for 7 mins
Also dont overdoo the dNTP's about 0.7mM is taken up in a typical reaction 
by the dNTPs so overdooing the nucleotides could be the problem.
Try a Mg tiration at 1.5mM/2.5mM/3.5mM and  dNTP titration of 1/0.5/0.2/0.1

but as chris said this is in any book or on lots of web sites.

Cheers
Bob (@home in bed with the flu since 28th december) Happy new year. :-)
> 1.1kb is small, not large and should present no problem for a compentent
> research tech.  You need to examine the purity and quantity of the template, and
> properties of the primers if things dont work.  We use Oligo 4 on a mac to
> examine the primer tm, binding properties of the primers....Usually if things
> dont work its easiest to redesign primers than to optimize.   If you want to try
> some quick optimization , try titrating the Mg conc.    I would suggest you do a
> web search using metacrawler, or mamma.com for pcr protocols... these are easily
> found.   Many web sites offer trouble shooting  guides for pcr problems  .   The
> companies that sell the Taq also offer tech support documents on their web sites
> that go into the many details of PCR.    There really is a lot of information on
> the web on this topic which you can find in about 15 minutes so i am not going
> into the details.  Usually the problem is that the primer design is not the
> best. If you are getting primer dimers this could because the primers bind to
> each other or the template is bad.  You will always get a product with pcr,,,,,
> but often not the correct one.   Without knowing more details of your problem it
> s impossible to trouble shoot it.
> 
> Scott C Winkel wrote:
> 
> > I am working on an amplification of a gene that is 1109 bp. This particular
> > assay has worked fine in other labs but has yet to be successful within our
> > facility. Other assays of smaller base pair length are working great, but
> > for some unknown reason this larger size assay does not want to cooperate.
> > Our primers have been checked and seem to be working fine. Actually , all we
> > seem to be getting are primer dimers. We are using new dNTP's, taq +Ab,
> > Mg++, & 10x buffer. The protocol is identical to what has worked flawlessly
> > in other labs. The only variables that are different are the technician,
> > location, water, and machine. We are currently using the Perkins Elmer 9700
> > thermocycler.
> >
> > If anyone has experience amplifying fragments this large I would love to
> > discuss the methods by which they are using.
> >
> > Thanks
> >
> > Scott
> >
> > scwinkel at prodigy.net
> 

-- 
Robert Hartley, Partick, Glasgow. Home of the RiscOS Molecular Biology page 
Home  r.hartley at freeuk.com    http://home.freeuk.com/r.hartley 
Work  rh at mblab.gla.ac.uk      http://www.gla.ac.uk/centres/cellengineering/





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