melting curve - (May/06/2008 )
hi guys,
i've done my first PCR and fortunately i don't have primer dimer or DNA contamination (NRT and NTC are negative). The problem is that my melting curve show 3 peaks, so i runned agarose gel and i see 3 band:
the expected band at 146 bp
another band more intensive around 190 bp, the Tm difference is about 2/2.5 °C;
the last band is around 300 bp
I've never done qPCR before.... what's wrong? Do i need gradient qPCR to optimize my primer?
Thanks a lot
i've done my first PCR and fortunately i don't have primer dimer or DNA contamination (NRT and NTC are negative). The problem is that my melting curve show 3 peaks, so i runned agarose gel and i see 3 band:
the expected band at 146 bp
another band more intensive around 190 bp, the Tm difference is about 2/2.5 °C;
the last band is around 300 bp
I've never done qPCR before.... what's wrong? Do i need gradient qPCR to optimize my primer?
Thanks a lot
It seemed you got non-specific amplification. YOu can try gradient PCR to optimize annealing temperature. What kind of template were you using?
I hope it's so!!! So I'll try gradient PCR.
I have another question... is it normal that the melting curve shows for one product (seen by agarose gel) different Tm?
I mean
gene X 85.8<Tm<86.2
gene Y 87.8<Tm<88.6
Thanks a lot
Your amplicon is about 146 bp. Because polymorphisms or mutations could happen inside the amplicon, you may get a range of melting point for that amplicon. As far as I have known, we dont have standard criteria to determine the Tm but an practical approach for in house sequence-independent Realtime PCR using SBGR is that you can use DNA/RNA of organism you want to detect in your own population to test your assay and then construct your own criteria such as average Tm +/- 2SD or 3SD.
Hope it is helpful!