I have a problem with my real time Dissociation curve. I got two peaks in all of my samples. I used the same primers that I used before and they were good (no any double peaks). Can it be some contamination in samples. its the first time that I got double peak in my real time dissociation curve.
Hey,
What you could do is:
order new primer (same primer code but maybe something is wrong with the primer mix)
measure sybr green not at 68°C, do it better at 80°C -> make a 4 step PCR:
95°C 15sec
60°C 15sec
68°C 15sec
80°C 15sec -> measure
repeat that cycle 40 times.
Unspecific signal should be gone at that temperature.
all the best,
Sebastian
Hi,
It can be a contamination from samples, if your NTC had any peak (which i cannot see that). Alos, if your negative does not displays any peaks in dissociation curve, the extra peak in your samples may be a template- dependent secondary structure from your primers pairs. as you mentioned, you've used before this primers, but did you tested on the same exact samples as you used now? If so, you better purchase new primers (samething on the primer mix, as mentioned by Sebastian).
Hope i could help.
Cheers,
Filipa
Dear Ketan,
I see you are getting a relatively large peak at 70. It is not small enough to be background noise, it's definitely a common sequence between samples that is being amplified. Having said that, I think it is rather exceptional to have contaminated all your samples simultaneously. I would check my primer mix first.
Additionally, negative controls are a must.
It might look like a primer dimer issue, but since the same sequence has been used before, I really doubt it unless you have significantly changed Tm.
I encountered this once, and could not determine the source until I changed the nuclease free water bottle, if you need to mix in most of your reagents like I do, make sure you check each separately.
Good luck!
Dear Sebastian, Filipa and Elia! thank you for your helpful suggestion. Regarding the primers I have used them a lot before and they are okay. I have even checked primer dimerization and they do not dimerize.
I couldnot change the thermal profile because we change it before. I even ran my old samples with this new samples together in same realtime PCR with same primers and same thermal profile. I didnot see double peak in my old samples but i got double peak in new ones.
From Elia's suggestion i think i used the leftover DEPC water from my friends tube. Maybe that caused the comtamination of my all samples.
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