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PCR

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.

duncan-bernard
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PCR

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  1. PCR

  2. 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. • Use a forward and reverse primer to start as the starting point and isolate the target DNA sequence.

  3. What do you need: 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.

  4. You will also need equipment: 1) The instrument that heats and cools a DNA sample for PCR is called a Thermal Cycler.

  5. Thermal block heats and cools to repeat the steps in PCR (20-50 cycles). 2. PCR tubes.

  6. Steps of PCR: 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!

  7. – PCR Thermal Cycling Parameters: Tina Doss Applied Biosystems 32 CYCLES 1 HOLD 2 HOLDS 95oC 95oC 72oC 72oC 10:00 0:15 55-65oC 0:40 10:00 0:30 4oC ∞ Denature Annealing Extension Final Extension Activation Final Hold This stage is to slow down all the processes and help keep the solution stable. This is like putting your sample in the refrigerator.

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