I still get Non-specific products & multi-peak Melt Curves despite changing the annealing temperature of my designed primers several times (58C, 59C, 60C, 61C, 62C, 63.7C & 65C).
I've also changed other parameters like the concentrations of DNA & primers, however, i still get these Non-specific products & multi-peak Melt Curves. Are there other factors that i can change to get rid of them?
Thanks in advance.
- Have you tried optimising your products by standard PCR (at different Tas) and running products on a 2% agarose gel in order to confirm specificity for subsequent qPCR
- In all honesty, if you are having recurring problems with multiple products at multiple annealing temp the best thing you can do is re design your primers
It shows that you have tried all permutation & combination, have to tried to change the primer stock itself??
Hello Sarah, how are you?
Your reagents are probably contaminated with other primers or DNA, I recommend you change it all. If you working with expression analysis, your cDNA sample has genomic DNA, which is amplified by your primers too. I recommend you design primers in different exons with at least 500bp intron in the middle. That will prevent genomic DNA that amplified in your cDNA sample. The tiny band appeared in the gel looks like a primer dimer. Your primer pair hybridize and amplified themselves. Maybe you need a new primer pair.
I hope it help solve your problem.
Best regards.
- Have you tried optimising your products by standard PCR (at different Tas) and running products on a 2% agarose gel in order to confirm specificity for subsequent qPCR
- In all honesty, if you are having recurring problems with multiple products at multiple annealing temp the best thing you can do is re design your primers
You can try to increase your annealing temperature if it is not a contamination.
Hi,
Need few additional details:
1. What is your target gene?
2. How is the amplification plot of Non Template Control?
3. Have tried Negative Blood DNA?
4. Which qPCR mastermix you are using?
Suggestions
1. Have you tried insilico analysis of your primers? Primer Blast?
This is help you identify potential dimers or nonspecific hits to human DNA.
2. Non-specific amplification can be avoided by qPCR buffer optimization, Titration of MgCl2 and the use of additives.
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