Hi,
I have attached images for the two questions I have. I use SYBR green for quantification. (a) I have my gene NF2 and I get a different melt curve for different samples. I get a single peak for rows D, F and G but in rows B, C, E and H there are two peaks irrespective of the sample. Is this a machine problem? (IMG_9435 and IMG_9436). This is the third time this is happening.
Secondly, my Ct values for NF2 are unusual and way lower than GAPDH. I have used the same sample in one row for GAPDH and NF2. So its not the sample. Can it be due to an error in primer dilution or some other error? (IMG_9437)
Thanks a lot!
Abhay Kanodia
I do not believe it is the machine. Genomic DNA contamination, maybe, depending where your primers annel. Isoforms, maybe also? I see it is triplicates, so it is your sample, all triplicates display the same pattern.
1)where you primers annel? exon-exon span? same exon? two different exons?
please use micromelt to see if it is expected two peaks in your melt curve https://www.dna-utah.org/umelt/quartz/um.php
if this is the case, run a gel to confirm https://www.idtdna.com/pages/education/decoded/article/interpreting-melt-curves-an-indicator-not-a-diagnosis
2)indeed very low Cq! Dilute your sample if your primers are okay.
Best regards,
Jacó
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