What is the possible reasons for getting this kind of melt curve in NTC (no template control)?

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Daniel Dehany Scott

Hi Lee,

Have you checked whether your primers are self- or cross-complementary (i.e. they are able to bind directly to themself or to each other)? It's possible that what you're seeing is products of primer dimers (given the successive steps and low melting temperature of these products).

There are several tools online to assess this, and you can also run your qPCR products out on a gel to see if you get low-Mw (30-50bp) products that are larger than single primers but smaller than your desired product.

Just one thought.

Good luck!

Dan

Boon Kiat Lee

Hi Dan,

Thanks for your suggestions. I've checked the primers complementarity and they have low self and self 3' (deltaG = -3 to -1) and no significant cross complementary.

FYI - I'm actually using the primers published by a paper

I did ran on a gel but I'm using a small gel and I ran almost near the end hence I might have missed out the low MW products if it happens to be there.

Previously I used this primer as well and the melt curve was not fuzzy as it is here. It is more of clean line with low melting point (60+). The difference between the previous run(1µl) and the current run (5µl) was volume of water added into the reaction mix.

Just a question for clarification, am I not supposed to get any signals or low melting point (60+) in my NTC?

Thanks

Lee

Hi Genghis,

I've actually used different sources of clean water and also tested by my colleague but the same thing happened to us (got the fuzzy melt curve).

Here is my reaction mix: 10uL 2X master mix + 0.1uL ROX + 2.8uL primers (0.7uM final conc) + 2.1uL H20+ 5uL cDNA = total 20uL. But if I loaded 1uL of H20 or cDNA, I will still have to top-up with H20 hence isn't the final concentration will also be the same dilution?

The PCR assay was optimised according to Taqman assay (the amount of water and cDNA loaded) but the master mix and primer concentrations are according to SYBR Green manual.

A question for clarification - is it normal if I get a melting peak for my NTC as I've gotten Tm peak for all my NTC(s) (60+°C). My melt curves for all my samples are single peak with a very minute bump at 60+°C.