User:Howard Boland/Notebook/Art from Synthetic Biology/2010/10/22

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PCR Optimisation
This protocol is a review to correct the PCR conditions from Monday based on Stratagen documents.

Mastermix PCR - pMAK512
 * 1) 80.2µl H20
 * 2) 8µl 10xpfu Polymerase Buffer
 * 3) 2µl pMAK512 plasmid template (100ng/µl)
 * 4) 2.5µl Primer forward
 * 5) 2.5µl Primer reverse

For each reaction add the following to each 50µl PCR tube
 * 1) 47.6µl Mastermix
 * 2) 0.4µl 10mM dNTP (not supplied with kit)
 * 3) 1µl pfu Polymerase

PCR conditions

Cycles: 30x Lid: 100ºC Volume: 50µl (each)


 * 1) Initial: 94ºC, 1 min
 * 2) Denature: 94ºC, 30 sec
 * 3) Annealing (Tm): 58ºC, 50sec (Lowest Tm 68ºC-10ºC)
 * 4) Extension: 72ºC, 1min 35sec (2 min/kb x 0.79kb)
 * 5) Goto 2, 30 times
 * 6) Final: 72ºC, 10 min
 * 7) Rest: 8ºC, forever

Mastermix PCR - pUA66katE
 * 1) 80.2µl H20
 * 2) 8µl 10xpfu Polymerase Buffer
 * 3) 2µl pUA66katE plasmid template (20ng/µl)
 * 4) 2.5µl Primer forward
 * 5) 2.5µl Primer reverse

For each reaction add the following to each 50µl PCR tube
 * 1) 47.6µl Mastermix
 * 2) 0.4µl 10mM dNTP (not supplied with kit)
 * 3) 1µl pfu Polymerase

PCR conditions

Cycles: 30x Lid: 100ºC Volume: 50µl (each)


 * 1) Initial: 94ºC, 1 min
 * 2) Denature: 94ºC, 30 sec
 * 3) Annealing (Tm): 56ºC, 50sec (Lowest Tm 66ºC-10ºC)
 * 4) Extension: 72ºC, 4min 43sec (2 min/kb x 2.359kb)
 * 5) Goto 2, 30 times
 * 6) Final: 72ºC, 10 min
 * 7) Rest: 8ºC, forever


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