Sorry wrong file

That was the melting curve for one of the first 2 samples then here is one of the amplification plots for the second 4 samples.

Primer dimers have a Tm lower than your PCR product, I don't think it's your problem. I currently have the same profile in my melting curves (unspecific peaks with a Tm higher than my specific peak while it was proved that my primers are very specific). I think that my problem comes from one of my primer stock solutions which degraded partially.

Another hypothesis would be that you used a too important quantity of DNA (genomic DNA or cDNA).

I'm waiting the others answers ^^

I had the same problem. I encourage you to test your qPCR amplification with SYBR green under different temperatures and using different combination of primer concentrations. This will help you in order to have the optimal amplification without primer dimer and strange peaks.

Could be that your cDNA conc is far far too high. Try 1E(-4) and 1E(-8) dilutions.

I should have clarified that this is genomic DNA not RT-PCR, thanks for your help.

Then just substitute "cDNA" by "DNA". I still think that a dilution may be beneficial.

Hi,

Too much DNA probably. Did you test your primers in qPCR before?

Doubble peaks in melting curve is not always something to worry about. Remember that this is measured after 40 cycles. By then your PCR is no longer quantetative, and you may have products produced at a late stage.

double curve could mean a variation in GC content, this is often due to heterozygous sample. the Tm normally shows a difference of no more than 3 degrees C

In the plot I can see it looks like your initial fluorescence is too high, which would result from too much DNA.

Also there appears to be no amplification at all.

Double peaks can occur when you have primer dimer as well as correct product. This usually occurs when you have only a very small amount of template. Be careful or you may be quantifying the primer dimer instead of the correct product. A way of getting around the dimer problem is to acquire data at a temperature above that which the dimer peak occurs.

Good primers are critical. Make sure the peaks are clean and high and amplification is very efficient. Otherwise you are always going to have problems

1. Regarding double peaks:

double peaks can arise in 3 common scenarios A, B and C

A. you have amplification of both primer dimers (peak with lower Tm) as well as the specific PCR amplicon (peak with higher Tm) in the same PCR reaction. This can occur (a) if primer design is not optimal (primer dimers will always accompany your specific amplicon no matter how much you optimize the PCR by varying primer conc. salt conc. annealing temp. etc) OR (b) insufficient template conc. (low abundant transcripts in case of RT PCR or poor quality degraded DNA in case of PCR) which gives sufficient time for your primer dimers to amplify before your specific amplicon. Remember that the more efficient your primer design, more quicker does your specific amplicon amplify...and as a result lesser chance for the primer dimers to amplify. An ideal primer for RealTime PCR should not form primer dimers even after 30-35 cycles in the absence of template (i.e. your NTC tube)

B. Your primer pair is amplifying two different amplicons (one which is specific and the other non-specific)

C. double peaks can also occur in case of certain PCR amplicons even in the absence of primer dimers. This is because certain PCR amplicons tend have dual melting domains. In this case both peaks correspond to the same PCR amplicon.

Scenario A cannot be used for RealTime PCR analysis as you are capturing the fluorescence of both primer dimers as well as the PCR amplicon.

Scenario B cannot be used for RealTime PCR analysis for similar reasons.

Scenario C can be used for RealTime PCR analysis and Ct calculations.

To confirm whether you have scenarios A, B, or C you have to perform agarose gel electrophoresis of the PCR product which had double melt peaks along with the NTC sample.

Heterozygous samples do not USUALLY give rise to double melt peaks in routine melt curve analysis. It might be apparent on High Resolution melt curve analysis.

I suggest you may try "Real Time PCR by Tevfik Dorak" which is an excellent read..

For the second question, it seems you have no amplification occuring OR you have not performed ROX normalization.

Kindly always include the agarose gel image while troubleshooting.

I prefer to use Syto9 green fluorescent dye (Invitrogen) over sybr green. It is far less inhibitory to the PCR reaction and gives more reproducibe replicates.