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PCR with Taq Mastermix
Source: Promega Usage Information for the PCR Master Mix (containing Taq Polymerase)
PCR Master Mix has been optimized for use in routine PCR reactions for amplifying DNA template in the range of 0.2-0kb
Protocol:
Thaw the PCR Master Mix at room temperature. Vortex the Master Mix and then spin it briefly in a microcentrifuge to collect the material in the bottom of the tube.
In a sterile, nuclease free PCR-tube mix following components for a 100µl reaction volume:
| Component
| Volume
| Final concentration
|
| PCR Master Mix, 2X
| 50 µl
| 1x
|
| upstream primer, 10µM (Bio seq f)
| 1.0-10.0µl
| 0,1- 1 µM
|
| downstream primer, 10µM (Bio seq r)
| 1.0-10.0µl
| 0,1- 1 µM
|
| DNA Template
| 1-5µl
| <250ng
|
| nuclease free water to final volume of
| 100 µl
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Guidelines for Amplification by PCR
- A. Denaturation:
- initial: 95°C 2'
- subsequent: 30 - 1'
- B. Annealing:
- 53°C 30 - 1'
- C. Extension
- 72°C 1'/kb of DNA to be amplified
- final: 5'
- D. Refrigeration
- 4°C (our thermo cycler can just go down to 12°C)
- E.Cycle Number
- 25-30 cycles
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