| | Re: calculating times & temperatures for PCR
There is no software I know of which would calculate time/temperature regime for your PCR reaction for a specific thermal cycler, given primers and lenght of your product. This can be done only theoretically and that calculation may even be unapplicable in a specific situation.
There are more factors responsible for a successful PCR. Each protocol has to be normalised, taking into consideration the type of polymerase, buffer and magnesium concentration, together with primers concentration of course.
There are some things recommended for start though.
1. If you don't have a gradient thermocycler then start with annealing temperature 5 degrees lower than primers melting temperature and consecutively increase this temperature to determine the optimal one - the one with no unspecific products and possible highest concentration of target product. (If you have a gradient thermal cycler then you're able to perform series of reactions with annealing temp. from-for example-50 to 60C at one go.)
2. It is generally considered that Taq polymerase binds 25 nucleotides per second on average (taking into consideration lower polymerase efficiency during last cycles due to thermal deactivation) at optimal temperature. This does not apply to long templates - time has to be extended. Calculate the time needed to elongate your target template, add time needed for your thermal cycler to reach optimal elongation temperature (the value of heating and cooling ratio in C degrees per second you should find in your manual) and add a few seconds to be secure. (Later on you can shorten that time to have more stringent reaction conditions - of course you can shorten the time untill you obtain your target product.)
It doesn't matter if you fall down as long as you pick something up from the floor when you get up.
Last edited by Aga; 03-04-2009 at 07:12 PM.