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-   -   PCR dimers - what does it mean? (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/84534-pcr-dimers-what-does-mean.html)

biochem123 01-29-2012 01:55 PM

PCR dimers - what does it mean?
 
I ran a gel on my PCR product. I have a lot of my product at the right size, but also high molecular weight dimers at 2kb. What could this 2kb dimer be possibly?

I know maybe it could be another site the primers could've annealed to and amplified, but since it is a dimer and not a single high molecular weight band, I don't think it is another annealing site for sure. Could it be single stranded DNA? If it is, why does it happen that the PCR generates single stranded DNA ? Thanks

admin 01-30-2012 12:29 AM

Re: PCR dimers - what does it mean?
 
Hi there if you post your gel result we can have a look but it sounds like your designed primers under the conditions you are using are annealing to themselves more than to your template / target sequence. This competes with the main reaction, and you aren't getting any high mw amplification.

Best thing to try first is to optimize the PCR reaction steps, first with annealing temperature - try higher annealing Temperatures or do a step down PCR.

Next you can always try designing new primers with low self-annealing properties. There are a few programs I used to do that check self-annealing.

davidflora 01-15-2013 05:26 AM

Re: PCR dimers - what does it mean?
 
[Only registered and activated users can see links. Click Here To Register...] Tm 40 is very low, so unspecific boosting and for beginners dimer is predicted. To fix this you need to improve the Tm, either by increasing the for beginners duration or by using LNA then improve you PCR


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