The primer annealing temperature , after the gradient PCR analysis, for the target gene differs from the optimal annealing temperature in housekeeping gene (positive control). What to do ? The difference is 3C, one primer is 57C and the other is 60C.The pcr protocol (syber green kit) says that the run should be run at 60C .
Hi,
depending on the used mix I would think the reagents are capable to compensate this difference. So I would run them a 60°C in a two step approach. Nayhow as you are only using sybr and not probe based Duplex you could run the reactions on separate plates but I would first give it a try with 60°C
Sven
Hi,
I suggest to do a touchdown PCR. If you can use PCR machine available with this feature. For the annealing temperature just set the highest temp for example 65 °C. Set the PCR to decrease the annealing temp by 0.5 °C after every cycle down to the lowest calculated annealing temp of your primers
(in your case 57 °C). Run the PCR at this temp for the following calculated cycle number. For example: (total cycle number)-(numbers of decreasing temp cycles) = (remaining cycle number). E.g.: 40 cycles - 20 cycles = 20 cycles at 55 °C. This technique is one step, compare to separate plate useage. It will maximize the amount of the specific product. It won't last longer than the original thermal profile duration.
Bests,
Tamas
I agree with Sven, the annealing temperature for primers is quite broad, in the past I have had even bigger differences in primer pairs and not run into problems.
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