PCR. What do you need: (the “master” mix). 1. DNA fragment to be copied 2. Nucleotide Triphosphates ( all four dNTP’s) 3. Primers (forward and reverse) need the 2 primers to “flank” the region of DNA to be copied.
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1. DNA fragment to be copied
2. Nucleotide Triphosphates ( all four dNTP’s)
3. Primers (forward and reverse)
need the 2 primers to “flank” the region of DNA to be copied.
4. Taq polymerase
(DNA polymerase isolated from bacteria-Thermus aquaticus, living in hot springs…their enzymes can withstand high temps!)
5. Reaction buffer – stabilize the pH
6. MgCl2 is needed for activation of the polymerase.
1) The instrument that heats and cools a DNA sample for PCR is called a Thermal Cycler.
Thermal block heats and cools to repeat the steps in PCR (20-50 cycles).
2. PCR tubes.
1)Heat to Denature (separate) DNA strands (95ºC) (~ 15 seconds)
2) Annealing: Cool to allow primers to bind (55ºC) (~30 sec)
3) Extension: Heat slightly so that Taq polymerase extends the 3’ end of each primer (72ºC) (~11 min)
4) Repeat steps #1-3 many times!!!
This three-temp cycle takes about 2- 5 minutes so 35 cycles will take ~3 hours to complete!
Thermal Cycling Parameters:
This stage is to slow down all the processes and help keep the solution stable. This is like putting your sample in the refrigerator.