I have designed 2 primers
Now I need to set up the PCR protocol.
First: I need to know how much of each primer, H20 and GoTaq® G2 Hot Start Taq Polymerase to put in the mix. We usually put a total of 14 ul in our PCR = 12 ul mix and 2 ul DNA.
Second: I need to know the temperatures and the durations and number of cycles needed to run the PCR in the thermocycler. Is there a rule to know that?
Please help .. Thank you
Talk with your research mentor.
You can calculate the volumes of each reagent based on the product information for your polymerase & the desired total reaction volume. Example: if the buffer is at 2X concentration, then you want half of the total volume to be from the buffer.
The exact times & temperatures for the PCR reaction will depend on the size of your product, the Tm of your primers, and the optimum cycling conditions for your enzyme. Again, check the product insert that came with the polymerase.
Again, talk with your research mentor. If they have a "usual volume total" for PCR, then they will have a standard protocol they use.
1- The annealing temperature depends on the nucleotide sequence of your primers (you can get it via many online tools: e.g. https://crm.vazyme.com/cetool/en-us/tmcal.html?)
2-The extension time of PCR depends essentially upon the length of the target DNA (amplicon size).
3- The cycle repetitions: 35-40 cycles
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