I dont know why im getting this kind of amplification curves. Anyone has an opinion respect to this issue ?
Christian Fernando Montoya Estupiñan By looking at your image are you using BioRad CFX96 PCR? PCR amplification is exponential in nature so you should see a sigmoidal curve not linear ones like yours. You can confirm by running your samples on a gel and check for PCR products. Try running a positive control. Start by making everything fresh make new working stock of your primers and probes, new nuclease free water, new master mix if possible, and add a positive control. May I know which fluorescent dyes you are using? Please also check your NTC.
Looks like you had no measurable increase in template. You should have a sigmoid curve, not a gentle positive slope. What did your positive control look like? Given that you've only highlighted a few of your samples, I'm guessing that the rest looked different. In that case, check your technical replicates & housekeeping gene to verify that you have cDNA from those samples.
Christian Fernando Montoya Estupiñan Please go for a melting curve analysis to make sure there are no misamplifications. It would be nice to do a primer efficiency test also, to me it seems like the primer efficiency is not good. Designing a new set of primers specific for your GOI should help.
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