I am currently performing qPCR and in the process of setting up relative standard curves to assess efficiency. I keep getting amplification in my NTC wells despite using new water and new SYBER green whenever I set up a plate. This leaves only the primers as a source of contamination. I use single use aliquots of primers (i.e. I throw away the remaining primers after setting up each plate). I used new water when diluting my primers and got TE buffer from someone who doesn’t even work in the same room as me or on the same organism. When resuspending my primers in TE I didn’t even use my pipettes (despite the fact that I have not even kept PCR product in the same room as my pipettes let alone used my pipettes with PCR product). Performing endpoint PCR and gel electrophoresis yields strong bands that are the right size for the amplicon (78bp) in the negative controls which appear to be different to primer dimers (which appear to be absent) (See lanes 4 and 8 in the attached images). One odd fact is that running a fairly high percentage gel (2%) for a longer period of time results in the separation of the band into three fainter bands (see attached images). Another oddity is that in several of the more diluted samples in my dilution series had Ct values higher than those of the “contaminated” NTC wells implying that there is less DNA in the more diluted samples than in the negative controls (which should be none). I am at a loss as to where this contamination comes from and where to go from here. Is it possible that this a some sort of artifact of the primers?
You don't have your target gene at all.
It's obvious.
So check your primers for blast, aneling tm and so on.
After that run a gradient pcr to reach target gene.
Regards.
Alireza Mordadi Thanks. What makes you say I don’t have the target at all? I didn’t design theses primers but I did BLAST them and they checked out (this PCR was run at a 55°C annealing temperature which is 5° lower than what they used in the article from which I got the sequence so am I right in assuming that one would expect amplification even if there is non specific amplification - as there appears to be in this one. )
Your positive control mad me to say it.
You don't have any band equal to positive control.
You can't do what you did for any primers specially those have low GC percentage.
Ok I see where the confusion comes in. The positive control is of a housekeeping gene to check that my mastersmix and cycling conditions were not completely wrong (it was included incase there was no amplification in my negative controls and would tell me whether this was because there was indeed no template or because something else was wrong with the PCR reaction itself (e.g. incorrect Taq concentration etc). Seeing as there was amplification this positive control serves no function. So that is to say, the positive control is not of the same target as the other gene (as would be the case if a cloned sequence was used with the same primers as is often done with positive controls) and so different sized fragements would be expected (the problem gene is 79 bp and the housekeeping fragment is 134 bp).
As for lowering the annealing temperature, (which is what I assume you mean by “you can’t do what you did“ ) running a PCR at the recommended temperature (and as determined by gradient PCR- see attached image) produces the same results (see attached image).
Yes. That was my point. Exactly 60 Celsius degree.
There is another question.
What concentration of cDNA do you use? And also about primers?
In the negative controls I didn’t add any cDNA and the standards are set up as a 10x dilution series with 8 steps. The qPCR was set up using 500nM primers but running an endpoint PCR with 1000, 500 or 200 nM all produce bands. 100nM does not produce any bands at all (negative control or otherwise) so it’s likely that this doesn’t produce enough PCR to be detectable by gel electrophoresis.
I recommend that you make the master mix with a new set of pipette s and template need to be added with another pipette in a different area or location at the end. This is a common practice to avoid contamination. Also
Also please check your products on a 3.5 per cent gel for proper resolution.
So I think you have your answer.
Con of primers.
There is no contamination.
Regards.
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