During my real time PCR experiment, I am getting a single peak at 85'C for even 42bp product. I am really surprising about this. Any suggestion for this?
Suppose, What your expecting? i mean to ask...at what melting temperature your expecting?
There is no surprise ....You may have primer dimers........or misanneling of your primer and amplifying different region
Do you see a melt peak for your negative control? If so, you have either primer dimers or contamination. Run a standard agarose gel and look at your products to see if you have primer dimers or unexpected sized products. 42 bp is a really small product, most reactions are optimized for about 100 bp products for real time PCR
Hi Balaji. Yes you correct to say the melt temperature is unexpected. However it is possible if it was GC rich.
Early on in the development of QPCR methods it is advisable to run products on a gel to confirm the product length is appropriate. I strongly suggest you do this now. It will work better if you make a gel with high agarose concentration, around 3%, and also make sure your ladder has a band at around 50BP.
I hope your primers are not overlapping - that could make a mess of it.
In my experience, I have always been dissatisfied with QPCRs that generated such short products. Usually efficiency is poor. And also sanger sequencing does not work on them.
If you continue to have problems I suggest you redesign the QPCR primers for a product of 100-200 BP, if that is possible.
Best regards
Roger Latham
Dr. Rajesh Parsanathan, your prediction may be right, but the surprising thing is I am getting a single clear peak at 85'C (which one showed previously at 75'C). If primer dimer or misannealing mean probably i might have get at least two or more peak. Thanks
Dr. Katie A Clark, I hope there should be contamination in my primer because even my NTC (non-template control) not good. Hopefully looking for getting new primers for this issue. Thanks
Dear Roger Latham, I have done primer efficiency for this, it was 99.93%, I have run the qPCR product in the gel it was good previously, now it is showing streak lane. The GC content is 50% only. I have done most of the trouble shooting by testing all the components used for the qPCR. I have done with another gene with 52bp its showing the exact place so the primers for this gene is the only problem, thinking of getting new primers, Hopefully it will solve this issue. Thanks.
Balaji it looks like you have already done some good investigation. That the QPCR has previously performed perfectly leads me to think about it differently.
A new batch of the same primers could be an answer, though unlikely if the old batch has previously performed perfectly.
Sometimes template has degraded, but if all versions of your template are showing the same erroneous peak then that is unlikely.
I would be considering a fault in your thermocycler - it's not as uncommon as you might expect. This week we had faulty thermocyclers at both institutions I work.
I suggest the following:
- Test your template with a different set of primers that definitely work well
- Test your primers with a few different templates
- Try another thermocycler
Best regards
Roger Latham
Roger Latham really appreciated. I have tried my template with another gene it works good, and another thermocycler, the result was same as I am getting. The clear conclusion is problem with primer so now gonna try with new set of primers. Thanks
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