I am currently trying to set up a gradient PCR to determine the optimal annealing temperature for my 16S primer pair. Our lab runs all our PCRs with the BIO-RAD T100 thermal cycler. This machine allows me to set the temperature range for my gradient PCR, which I have set to 60-65oC. However, the temperatures in between are not increasing in equal intervals. Instead, the temperature interval increases before decreasing as the higher temperature limit is approached
What I want to know is the following:
- Is it important that the gradient remains equal (temperatures increase at equal intervals) when conducting a gradient PCR?
- Can I make the intermediate temperatures increase at an equal rate? If so, how?
Thanks!
There always not equal intervals between rows when performing gradient PCR using T100 thermal cycler. If you want to test several temperatures for your primers, you just need to play with temperature parameters of the gradient PCR program. If you want to test 50 degrees but machine displays 50,1 it is ok, just go ahead. If temperature is too critical for you then you need to set up several PCR reaction one by one or use several thermal cyclers.
Gradients are (1) set for a range say 45-65 and the instrument auto calculates for the 8 or 12 rows of your PCR slots for which you would obtain the values of tempratures used in each well or simply (2) run PCR with 5 C increment starting at 45 C until 65 C or so- basically 5 reactions to see results- if at all. Any PCR machine can do (2). best wishes.
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