Could anybody help me with my qPCR issue? I have cellular gDNA from human PBMCs serve as my target gene negative control. I did blast and in silico test, my qPCR product do not have any match with human genome. However, my negative control sometimes give positive results. When I check the amplification curve, it turns out the negative control always have a plateau phase plus a amplification phase in the end of the curve. I test this primer set in SYBR green qPCR, and got exactly the same amplification curve. The melting curve showed a specific peak same as positive control in combination with a small unspecific peak. Since the water is completely negative, dose this means the primer sets could specifically pick up something from human gDNA?
Yes, it seems your primers pick something up. I would also do what Laurence suggests. I used to order always a couple primers that were shifted by 1-2 nts, so that I could run several possible combinations. Often, the best pair I found empirically was *not* the one proposed as optimal by the software. Ordering new primers is usually faster and cheaper than optimizing reaction conditions to make suboptimal primers work (sufficiently) well.
Thanks a lot for your reply! Indeed, I tried more than 5 sets of primers. Two of them were published in literatures also. With all of them I got the same problem. Would it be the contamination of my negative control? I tried seral different DNA isolations and really precaution with all kinds of contaminations. Would it be possible from the aerial contamination?
Being published does neccesarily not mean being good...
The negative template should not be a problem. The primers should be specific in any case,
You could try different master mixes (suppliers, brand), if you have already tried differrent primers.
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