PCR Optimisation
This protocol is a review to correct the PCR conditions from Monday based on Stratagen documents.
Mastermix PCR - pMAK512
- 80.2µl H20
- 8µl 10xpfu Polymerase Buffer
- 2µl pMAK512 plasmid template (100ng/µl)
- 2.5µl Primer forward
- 2.5µl Primer reverse
For each reaction add the following to each 50µl PCR tube
- 47.6µl Mastermix
- 0.4µl 10mM dNTP (not supplied with kit)
- 1µl pfu Polymerase
PCR conditions
Cycles: 30x
Lid: 100ºC
Volume: 50µl (each)
- Initial: 94ºC, 1 min
- Denature: 94ºC, 30 sec
- Annealing (Tm): 58ºC, 50sec (Lowest Tm 68ºC-10ºC)
- Extension: 72ºC, 1min 35sec (2 min/kb x 0.79kb)
- Goto 2, 30 times
- Final: 72ºC, 10 min
- Rest: 8ºC, forever
Mastermix PCR - pUA66katE
- 80.2µl H20
- 8µl 10xpfu Polymerase Buffer
- 2µl pUA66katE plasmid template (20ng/µl)
- 2.5µl Primer forward
- 2.5µl Primer reverse
For each reaction add the following to each 50µl PCR tube
- 47.6µl Mastermix
- 0.4µl 10mM dNTP (not supplied with kit)
- 1µl pfu Polymerase
PCR conditions
Cycles: 30x
Lid: 100ºC
Volume: 50µl (each)
- Initial: 94ºC, 1 min
- Denature: 94ºC, 30 sec
- Annealing (Tm): 56ºC, 50sec (Lowest Tm 66ºC-10ºC)
- Extension: 72ºC, 4min 43sec (2 min/kb x 2.359kb)
- Goto 2, 30 times
- Final: 72ºC, 10 min
- Rest: 8ºC, forever
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