no increase in fluorescence in my real time PCR
Posted 02 February 2011 - 07:51 AM
I have a trouble in my real time pcr runs. The gene that I have interested in does not work in real-time pcr. I have optimized the conditions for the primer in PCR before, I have tried both genomic DNA and cDNA as template and observed nice bands on the gel . However, when I have switched to qPCR I couldn't observe any increase in the fluorescence (using cDNA as template). Also I have loaded qPCR products onto gel and as expected I could not observe any bands.
I am using FastStart SYBR Green Master from Roche and the machine is Rotor-Gene 6000. While my reference gene works very well in qPCR runs , my gene does not. I have tried different dilutions ; first I've tried 1/20 & 1/200 dilutions. Then thinking that the template might be too dilute I've used more concentrated cDNAs (no dilution & 1/5 ).
Icould not find an explanation for this situation. Do you have any suggestions or possible explanations?
Thanks a lot:)
Posted 02 February 2011 - 08:20 AM
Posted 02 February 2011 - 11:28 AM
Thanks a lot for your suggestion.I want to mention that my primers are working just fine with RT-PCR and they are also appopriate for qPCR, product size is 132 bp (not so big). Therefore, I think my primers are ok.
Posted 03 February 2011 - 05:20 AM