Hi every body,
I want to set up real time PCR for one gene, but after doing it I get two or three peaks in melting curve of real time PCR. The designed primers for my gene is very specific. After dilution of cDNA, the extra peaks dropped but did not disappear.
How can I decrease or remove extra peaks and set up real time PCR? The picture of gene melting curve is attached.
How can I calculate melting temperature of my amplicon? I know that it depends on GC percent of amplicon. Is there a site for calculate the melting temperature of amplicon?
Please guide me.
Thank you.
https://eu.idtdna.com/calc/analyzer
Additional peaks could include non-specific amplification, i.e. as a result of genomic contamination among other things.
However it could be a benign difference in GC-rich regions melting as DNA melting Is not always biphasic (see page 22 of attachement).
I doubt it's a primer dimer/self/hetero dimer issue as the secondary peak at 88Tm suggests either a bigger amplicon than the primary one at 84Tm or it is more GC rich.
A gel image would give a more definitive answer.
https://eu.idtdna.com/calc/analyzer
Additional peaks could include non-specific amplification, i.e. as a result of genomic contamination among other things.
However it could be a benign difference in GC-rich regions melting as DNA melting Is not always biphasic (see page 22 of attachement).
I doubt it's a primer dimer/self/hetero dimer issue as the secondary peak at 88Tm suggests either a bigger amplicon than the primary one at 84Tm or it is more GC rich.
A gel image would give a more definitive answer.
Dear Can, Hassan and Khalid, Thanks for your very good comments. Dear Can, Thank you for sending melt curve file. The gel picture is attached. The well (Lane) 1 is related to desired curve. Best regards, Ramin
There appears on lane 1 two distinct bands suggesting two seperate amplicons. Any chance you can share the primer sequence?
Dear Can, The sequence of my primers is as follows:
F: ATTCTCAGTGGAGACCGCC
R: GCTGTGAGGACTCAGGGTG
I am assuming you are working with mouse, if not let me know which species. I did a BLAST analysis of your primer and found that your reverse and forward primers are distinct. You forward primer has the potential to bind to several splice variants, but this is a prediction and these isoforms may not even be expressed in your tissue/cell. You may want to double check the reverse primer sequence because it is completely unrelated to the forward. See the ppt. attachment for results.
https://blast.ncbi.nlm.nih.gov/Blast.cgi
Dear Can, I am working on human samples.
Thanks for your comments.
Best regards,
Ramin
It looks like your primers are spanning 4 exons, so you may be amplifying more than 1 exon. What is your buttom 3 ladder sizes from the gel image? What was on lane 2, 3 and 4? Different primers?
Dear Can, The forward primer is designed in exon-exon junction, and the amplicon size of this gene is 118bp. Thus, the designed primers are spanning 2 exons. The used ladder is 100bp-3K.
Lane 1 and 2 was desired gene. Lane 3 and 4 was GAPDH gene (Amplicon size: 107bp).
Best regards,
Ramin
Good morning Ramin.
I have spent months and months resolving this sort of issues during my PhD and here is what you are seeing.
Your primers are amplifying more than 1 product. This is clear from your agarose gel picture. There are 3 bands representing 3 separate products.
This is due to the primers binding in other locations and not just where you have designed them to bind.
Using NCBI BLAST will allow you to identify what are the products of you primer pair. Would highly recommend starting from there.
If you will find that your primers are amplifying other genes, your best bet would be to change the location of your primers slightly and do BLAST on those again, until you will find no more additional products.
you could try getting rid of the additional bands by increasing the temperature of your primer binding step in the PCR reaction. But that does not always work that well.
Another issue, which is very rare is that your primers may be binding to each other and forming these extra products. But that is rare.
To check your primer properties such as melting temperature, ability to form primer dimers etc, try the IDT technologies web site then go Tool and Oligo Analyser tool. Paste your primer sequences there and see what output it will come out with.
Hope this helps
Hi dear Yegor, Thanks a lot for your comments. Thanks for your kindness. Best regards, Ramin
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