RT PCR strange melting curve - (Feb/22/2007 )
I am working on real time PCR with Sybr Green. My aim is to develop stable real time assays to quantify different functional genes from environmental samples (soil). As a very beginner in real time technique I have some troubles to interpret my results... and thus to react on what I see. I did rt for a 630bp gene, and my first question would be, which effect has the size of the fragment amplified (I read somewhere in this forum it should not be bigger than 400bp..), what are the criteria for this? Efficiancy was rather ok, but the melting curve looked strange for the Standards (in green) but not for the unknows. What could be a reason for this? Negative controls (in yellow) could not be detected on agarosegel, am I right to assume this is primer dimer?
Hoping for help!
I think you might see a shift like that in the positive band due to a single bp (ie: SNP) difference between the amplimer from standards and unknowns.. You mentioned you ran a gel was there one band of the correct size in each amplification? I agree the yellow is primer dimers. To prevent primer dimers from affecting your sybergreen assay you can do denature, anneal, extend, goto 81 degrees C for 2s and do a single acquisition read of sybergreen --that should melt off your dimers and prevent them from looking like positive samples during amplification. Please note that this is not as good as having primers that do not form dimers because you still have primer dimer formation which affects your efficiency etc, you are just manipulating the data collection to ignore those products HTH--Good Luck!
Thanks for your advise! Yes, the bands had the correct size. I tried this single acquisition read with real time (I hope I got it right that you meant meassuring after the melting point of the dimer)with another gene (about 450bp) ( at 84°C, because this was the melting point of the primer dimers, products where melting at 91°C) and got kurves that did not reach the plateau but seemed to decrease. Would you also have a suggestion for this? Was this temperature too high?
I don't know, never tried with a higher temperature, are you sure you had primer dimers that melted at 84C that seems high for as short the dimer sequences should be...