A DNA sample of 2000 base pairs was subjected to PCR under the given conditions Initiation; 950C for 5 minutes Denaturation: 950C for 45 seconds Annealing: 550C for 45 seconds Elongation: 720C for 120 seconds Final extension: 720C for 10 minutes How much time does it take to complete 35 cycles?
I tried to calculate it as follows : [35 * {300+45+45+120+600}] which comes out to be 11,310 sec or 188.5 min. But the answer comes out to be 137.5 min Can anyone please rectify my mistake? I think the extension time should not be in the cycle and should be considered in the last cycle. is it true?
Hi Vinay,
The right calculation formula is: 5min+35(45sec+45sec+120sec)+10min=137.5min, only pcr is repeated 35 times, preliminary denaturation and final extension are not.
The above calculation by Aïssa Saidi would be correct except for the fact that it doesn’t take into account the time of ramping up and down the temperature between the different steps. This will extend the total time of the run by a duration that depends on the thermocycler and on the reaction volume.
I agree with you dear Pierre, absolutely, I neglected a few second fractions of the ramping time.
I totally agree with you Pierre Béguin Sir. So for here can we consider the pre-heating of the thermocycler to 1000 C and then when it reaches to 950 C we started the rxn. Is this valid ?
Exclude the initial denaturation and final extension from the 35 cycles,, those two are just once in the PCR
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