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Rifat Rumki

Member Since 28 Sep 2011
Offline Last Active Feb 22 2013 08:28 AM
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#120779 Unable to PCR!!!!

Posted phage434 on 03 October 2011 - 04:50 AM

I would check that my template cDNA was good by trying to amplify another gene.  Although those cycling conditions look as if they might work for Phusion, they will not work for Taq or many other enzymes.  Taq will be degraded at 98.  Your reverse primer has a very high GC content at the 3' end, which may cause non-specific priming.  You are likely adding too much primer (100 pmol vs. recommended 25 pmol).  You could try adding 3-6% DMSO, but my first recommendation would be to redesign the primers, probably by adding a few bases at the 3' end.  High marks for providing lots of information to us.


#120720 Help designing primers with restriction sites

Posted phage434 on 01 October 2011 - 03:36 PM

I think the primer region binding to your template is too short.  That is what determines the Tm for early cycles, so it needs to be longer.  You have only 13 bases, while a typical good primer is 18-22 bases long in the template binding region.

Also, given a choice, I would not choose BamHI as an enzyme, since it cannot be heat killed.


#120193 PCR problem

Posted allynspear on 22 September 2011 - 11:17 AM

First of all, when you are describing a reaction set up, you need to specify concentrations, not volumes.  For instance, if you are using a standard 10mM dNTP stock, then 7 uL of 10mM dNTPs is way too much.  Usually 1 uL of a 10mM dNTP stock in a 50 uL reaction (0.2 mM dNTP final concentration) is used.  The same with primers.  Each polymerase is different, but a typical range would be 1 uL of a 10mM primer stock in a 50 uL reaction (again, 0.2 mM final concentration).  Lastly, your template (miniprep DNA) should be at a final concentration between 0.1 nM and 0.5 nM, which means that for an average 3000 bp plasmid, you would include about 50 ng of DNA in a 50 uL reaction.

All of these concentrations can affect how well your PCR works.  Lastly, your PCR cycling conditions are also critical.  As Adrian K suggested, you could try gradient PCR to find a good annealing temp, but in case you don't have a gradient PCR machine, I would just try this PCR program:

95 degrees, 2 minutes
----------------------------
35 cycles of:
95 degrees, 30 seconds
50 degrees, 30 seconds
72 degrees, 1 minute/kb of PCR target
-------------------------------------------------
72 degrees, 10 minutes
4 degrees, infinite

This program may generate some non-specific products, but you should at least get the band you are looking for.  If you need to get rid of backgroud, you can always try raising the annealing temperature (from 50 to 55 or 60) AFTER you can actually get a product.

Best of Luck.


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