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PCR troubleshooting: wrong amplicon size when spiked into sample - (May/13/2014 )

Hoping I can get some help here. My graduate student is using real-time PCR with an amplicon of 200bp. Her positive control is the correct length and melting temp but when she spikes already extracted DNA into extracted matrix her amplicon is only 100bp. Any ideas of why this might be occurring?





You might want to use Klen Taq

-Matt Tan-

First some questions. I really don't have an idea what are you trying to do and why.



What are you detecting by real-time PCR? Fusion gene, knockin gene, some variant,...? 



The use of positive control spiked into sample is only used for inhibition control after failed PCR or as an internal control in all samples, which is then required to by of different lenght/melitng/dye color etc.



What is the positive control? Plasmid DNA, PCR product, gDNA of "positive" something?



What do you mean by "already extracted DNA" extracted from what?



What is "extracted matrix"?


The PCR target is the ITS region of a parasite of cervids. The disease is vector borne by horse flies. We started exploring this situation when low Ct values in the real-time platform corresponded to the wrong M.W. when run on a gel. We are testing extracted fly heads as our matrix for the presence or absence of the parasite. When we got the wrong M.W. we were looking for inhibition and noticed that our extracted positive control parasite DNA spiked into extracted fly heads was still coming up with the wrong M.W. The positive control was from whole adult worms and came was the correct size on the same gel (without spiking). Hope that helps but please let me know if I can provide more information.


I see.


You are running SYBR assay on this I suppose.


If you are getting any amplification, it AFAIK not an inhibition. More likely an unspecific product. It's possible it preferentially amplifies even in the presence of you positive control.

I also encountered primer dimers (that would fit in the size) that were behaving inconsistently, not present with specific template and present in the NTC or so. You may have a case of such weird dimers, meaning your sample is actually negative.


I would say, that with the heads only you get 100bp, and with the heads and positive control you also get 100bp, that there are no differences between these two amplification, but I don't have and idea why the positive control would not amplify and this weird band would, but it is a possibility.


You may try to sequence the 100bp band, but it's quite short to get anything useful.


Do you have any other PCR assay to test on fly heads that there is no inhibition? (some fly DNA assay)

If you suspect inhibition, other way how to test is dilute the template DNA 10x, 100x, 1000x and so. And look if it changes anything in the product size or enhances the amplification.


I suppose possibility, that there may be a variant of the ITS region that is shorter is ruled out.