pcr
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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.
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.
You will also need equipment: • 1) The instrument that heats and cools a DNA sample for
PCR is called a Thermal Cycler.
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!
– PCRThermal Cycling Parameters:
95oC
10:00
95oC72oC 72oC
55-65oC0:15
0:30
0:40 10:00
4oC
∞
1 HOLD 32 CYCLES 2 HOLDS
Activation Denature Annealing ExtensionFinal Extension
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.
Tina Doss
Applied Biosystems