I am using Arabidopsis thaliana genomic DNA sample to amplify telomeric sequences by qPCR. However, my primes seem to amplify themselves, so I see Ct values for my NTCs. I'm almost sure there is no contamination because I renew everything over and over. Now I would like to first try to amplify the telomeric sequences with conventional pcr, then use these products as my template for qPCR (using the same primers). Is it possible or acceptable? Any other suggestions? Thank you.
Dear Zeynep,
In general, your strategy is ok, but, there might be some technical problems. For accurate and reliable qPCR quantification, your qPCR amplicon is supposed to be short - about 100 bp. But your possible primer-dimers are also short, so you should be careful during the gel purification of your PCR product - avoid contamination with the primer dimers. The best would be to use 2.5% agarose gel and a 50-bp DNA ladder for running your PCR products. After running the gel, cut the PCR product out of the gel, purify it using an appropriate gel-purification kit, measure DNA concentration, and prepare 5 standard dilutions (e.g. 4-fold each step) of the purified PCR product. Use these standard dilutions to make a standard curve for your qPCR.
Adding to the answer Pavel Uvarov provided. There are purification kits that will remove the primers from your PCR product without doing a gel extraction. I have used the Monarch PCR & DNA Cleanup Kit from NEB with good results.
If you are able to design new primers, that is probably worthwhile. There are tools that will predict whether your primers are likely to form homo- or hetero-dimers. I always check my primers using IDT's free OligoAnalyzer tool before ordering to confirm that they will perform adequately.
Hi Pavel, this is possible but I would use new primers that are more shifted in the middle of the sequence (see also nested pcr).
Best regards, Anna
Hi Anna-Maria Hartmann, you are absolutely right regarding the nested PCR strategy. Indeed, the best would be to order a new primer pair for the amplification of a larger gDNA fragment (e.g. 500 bp) that contains binding sites for the existing qPCR primers. Purification of the 500 bp fragment should be easy, and the purified 500 bp fragment can be used for the optimization of qPCR conditions with the existing primers.
Thank you all for your answers. I guess I can't use the nested PCR strategy because my target is telomeres and they have the same repeats?
I need to see a smear band for my sample due to the different sizes of telomere sequences being produced. I got what I needed on the gel with the PCR after PCR, but I still see peaks for NTCs. Also one of my samples gave negative RFU. Maybe I need to dilute the template? (Red peak is NTC)
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