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