Better efficiency but bad melt curve - (Mar/09/2006 )
Hi!
To optimize my PCR efficiency, which was too high yet (120-140%), I decreased my qRT Gapdh primer concentration from 25pmol to 15pmol and leaved out the elongation cycle, because annealing time was already high (30sek) and the elongation step did no further elongation and used QUIAGEN isolated plasmids for standard curve.
Now the efficiency is nearly 100% which is great, but my melt curves changed, as you see on the pic. So do I have unspecifc products now for the lowest dilution now? 10^6 to 10^0
Thanks for your help, Sera
-Sil-
QUOTE (Sil @ Mar 9 2006, 06:35 PM)
Hi!
To optimize my PCR efficiency, which was too high yet (120-140%), I decreased my qRT Gapdh primer concentration from 25pmol to 15pmol and leaved out the elongation cycle, because annealing time was already high (30sek) and the elongation step did no further elongation and used QUIAGEN isolated plasmids for standard curve.
Have you linearised the Plasmids before?
This ir really important!
Now the efficiency is nearly 100% which is great, but my melt curves changed, as you see on the pic. So do I have unspecifc products now for the lowest dilution now? 10^6 to 10^0
Thanks for your help, Sera
To optimize my PCR efficiency, which was too high yet (120-140%), I decreased my qRT Gapdh primer concentration from 25pmol to 15pmol and leaved out the elongation cycle, because annealing time was already high (30sek) and the elongation step did no further elongation and used QUIAGEN isolated plasmids for standard curve.
Have you linearised the Plasmids before?
This ir really important!
Now the efficiency is nearly 100% which is great, but my melt curves changed, as you see on the pic. So do I have unspecifc products now for the lowest dilution now? 10^6 to 10^0
Thanks for your help, Sera
-Montgomery Burns-