I would like to know what happened to one melt curve that I get from qPCR. Among 6 products, only one this product showed such melt curve. I repeated again and also gave me the same one while others were ideal peak as those should be.
Can I say non specific product? If non-specific, how much vary from expected target product? Just small different or How? Thanks
Hey Jamail,
looks like you have 2 PCR-products caused by an amplification of a second primer-target. The easiest way to be sure is to load your PCR-products on a 2% agarose gel and check the bands.
The melting point can give you a hind about the length of your products, but it also depends on the sequence.
To avoid unspecific binding you can try several things:
1. BLAST to check for similar sequences
2. Change the sequence of your primer (use BLAST to check for unique sequences)
3. Increase the annealing temperature (minimizes the risk of incomplete primer binding)
4. Reduce the concentration of your primer (but check the efficiency afterwards...if it works)
Good Luck!
Maybe your target sequence contains a sequence variation (SNP)? The primers would be locus-specific, and yet there will be more that one product sequcence amplified, what can possibly lead to slightly different Tm's. A search in sequence databases can give you a hint if this is likely at all.
You could run a native 10% PAGE of the PCR product and see if there is omer than one band. If you see a single band, it is most likely that there is a base exchange. Two bands that differ only by 1-3 bp may indicate some short ins/del variation. Larger differences make it more likely that you are amplifying in fact different loci.
I think you will only get a final anwer when you sequence your PCR product.
Another idea to check if these are SNPs:
Dilute your sample so that one PCR reaction contains an average of at most 1 target copy. You should then get about 60% positive PCRs, each one having a single product with a Tm of either 83°C or 84°C.
same primer amplifying with another short gene, standardize different annealing temperature, you will get the normal melting curve.
Good luck
I would like to know available online software that suggests to add 6 nt base pair (ATCG) at 5' end of my primer in order to become primer Tm to 60 degree -/+ 1 for qPCR. One suggested to use idtdna.com/analyzer but I am not sure how to use that one. Are there any better way to achieve this? Thanks.
Adding such 6 nt to my primer will not affect my amplicons in qPCR.
Thank you guys. Finally I got nice cure. The issue was that one nucleotide was wrong. I corrected it and finally got this nice curve.
I have another one issue. Pls see next question that I am going to post. Pls send me your precious advice. Thanks.
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