Dear All,
I have been working with a set of primers which was designed a particular cDNA for over a year. For over few weeks now, I find the same amplicon size (0.4kb) in any and every sample; exception being genomic DNA of my model plant and other Bacteria with which I find no amplicon at all. I have tried to increase the annealing temp to 60 degrees, but the band only gets brighter. The water control shows the brightest. I have tried ruling out the stock being contaminated by using primers directly from the Stock Primer Tubes. Are my primers contaminated? Or is there any other explanation as to how I find the same band in every PCR? Is there any other solution to solve the problem other than ordering for new set of primers?
Please help,
Thanks...
Hi Sharon
I dont quite understand your question. Do you want to provide more details then I might be able to help :-)
hiiii sharon..... It can happen....I will suggest you to change all reagents once including yours water source...pippetman as well and restart your work in a laminar flow cabin.
If you are getting amplification from water of a 0.4kb fragment, your primers are contaminated, or your PCR reagents are. If this is not happening with other primers, the contamination is in your primers.
Sounds like a classic case of amplicon contamination of some PCR reagent. Since the final amplicon is an extremely concentrated "template" for PCR, it does not take much of it to cause rampant contamination. And if you are working constantly with the same product, you have to be extremely careful to keep all "post-PCR" product away from the reagents and equipment that you use to set up PCR reactions. The only mystery is why your water control is giving you a product band, but some of the other DNA templates that you mention are not. Maybe just coincidence--if you set up 10 water controls, for example, you might only see product in some of them if the contamination is at a very low level.
Be sure to use a new sample of polymerase too (not just the other consumables). If you're really curious, you could sequence your mystery band.
If your water is the source of the contamination, then it would be worth making small aliquots of water just for PCR and using a fresh one each time.
Dear All,
I have tried and tested all the advices above. I even ordered for a new set of primers and performed the PCR just to find the band intact, even in my water control! I have started using DEPC treated water. I even changed my work bench twice, in different LAFs; but the band is still intact. Any more suggestions?
Thanks in advance!
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