This particular qPCR melting curve (refer the attached) has issue on its left side of the curve as it was a big higher. Ran 3 times with same qPCR profile and got 3 different melt curve as you see in the attached file.
Curve 1 is not that bad but as mentioned a bit high on its left side.
Curve 2 and 3 showed that the high one on its left become amplified too.
So what should I do with this. I did change my primers design in two different ways. However, still come out the same curve too. Kindly post your advice. Thanks.
Hi Jamail,
If you run your samples on a gel do you get two bands?
You could possibly have primer dimer, which means you need to adjust your annealing temperature (has this been optimised for you new and old primers).
The second thing it could be is contamination. What does your non template control curve look like? I would change you stocks, clean your workspace and pipettes and try again.
Cheers
Is it possible for your target gene to be present in two copy?
First, run out your PCR products on a gel to see if you have only one product.
This is NOT primer dimers - they will melt at a low temperature.
It's probably not contamination, unless you see a peak in your negative controls.
If you have a large sequence AND/OR a short sequence that has regions of two different GC content, then you can get a product that melts in more than one phase. This will give you a "jagged" peak like you are seeing.
As long as you can verify that you have exactly one PCR product (agarose gel) and your controls are all working, then you simply have a product that melts in stages. Totally normal.
Dear Jamail,
The reason for getting 2-3 peaks in your QPCR could be many. Excellent suggestions were given, i will give some from what i know.
Firstly when you mean qPCR i assume your are not looking at RNA expression, if you are the
1. make sure your primers are exon - exon spanning
2. standardize your annealing temperature
3. Make sure the RNA has not degraded
If your are only doing QPCR
1. BLAST your primers and check whether it gives unintended products and see their size, the primers may be amplifying several products
2.The agarose gel may not be able to resolve PCR products of close size, in that case melt curve is ideal, so if your melt curve gives you two peaks then you need to sort out the issue, running products on agarose gel may not help
3.Like Sarah said, contamination is an issue, make sure the DNA is not getting contaminated especially while using pipettes and tips, tubes.
4. Also make sure you are using primer in the range of 100-400 uM, too much of primers can sometimes give you such peaks.
5 Also check your using the correct SYBR green (with or without ROX) in case of SYBR green or Eva Green. Check the compatibility of your PCR mix with your machine.
Hope you sort out the issue. To me its most likely that your primer is amplifying two products.
All the best.
Regards,
Prasad.
Larger PCR products almost always give this sort of jagged peak. So will reactions from heterozygous insertion/deletion alleles. You could always clone and sequence to see exactly what you are amplifying.
Hello Guys, Thank you for all of your advises. Finally, it was solved. The issue was one nucleotide in primer was wrong for this case.
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