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PCR Protocol, PCR Steps
PCR protocol, PCR steps
PCR protocol
In a traditional PCR protocol, reaction components are assembled as described below. The final volume should be 50 µL.
- Thaw all reagents on ice.
- Assemble reaction mix into 50 µL volume in a thin walled 0.2 mL PCR tubes.
- Add reagents in following order: water, buffer, dNTPs, Mg CL2, template primers, Taq polymerase.
- Gently mix by tapping tube. Briefly centrifuge to settle tube contents.
- Prepare negative control reaction without template DNA.
- Prepare positive control reaction with template of known size and appropriate primers.
- Program your thermocycler for your PCR reaction using the following guidelines:
- Analyze the results of your PCR reaction via gel electrophoresis.
Component |
Final Concentration/amount |
---|---|
water |
to 50 µL |
buffer |
1 x |
Taq polymerase |
0.05 units/µL |
dNTP mix |
200 µM |
MgCl2 |
0.1-0.5 mM |
Forward primer |
0.1-0.5 µM |
Reverse primer |
0.1-0.5 µM |
template |
200 pg/µL |
DMSO (optional) |
1 to 10% w/v |
PCR steps

Step | Temp | Time | # of cycles |
---|---|---|---|
Initial Denaturation |
94°C |
5 min |
|
Denaturation |
94°C |
30 sec |
30-35 |
Primer Annealing |
Tm-5°C |
45 sec |
|
Extension |
72°C |
1 min per kb |
|
Final Extension |
72°C |
5 min |