User:Howard Boland/Notebook/Art from Synthetic Biology/2010/11/03

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PCR Product, 2.3kb product

After Saturdays unsuccessful PCR attempt, I set up 4 new tubes each using different template and paying particular attention to pipetting.

Master Mix

  1. 160µl H2O
  2. 16µl 10xPFU Buffer
  3. 5µl Forward Primer
  4. 5µl Reverse Primer
  5. 2µl dNTP
  6. 4µl PFU DNA Polymerase

Aliquote into each of 4 tubes

  1. 48µl Master Mix
  2. 2µl DNA Template
    1. Tube 1: pUA66katE 25-10-10-BLK
    2. Tube 2: pUA66katE 25-10-10-NA
    3. Tube 3: pUA66katE 30-10-10
    4. Tube 4: pUA66katE, colony (10µl H20)


PCR conditions

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

  1. Initial: 94ºC, 1 min
  2. Denature: 94ºC, 30 sec
  3. Annealing (Tm): 56ºC, 50sec
  4. Extension: 72ºC, 4min 37sec (2 min/kb x 2.359kb)
  5. Goto 2, 30 times
  6. Final: 72ºC, 10 min
  7. Rest: 8ºC, forever

PCR Product, 2.3kb product

Result of PCR 1% Agarose Gel, 03-NOVEMBER-2010-10:44 GMT

  1. Lane 1: 10µL 1kb NEB Quick Ladder
  2. Lane 2: 50µl PCR product, pSense66 (2300bp), 25-10-10-NA
  3. Lane 3: BLANK
  4. Lane 4: 50µl PCR product, pSense66 (2300bp), 25-10-10-BLK
  5. Lane 5: BLANK
  6. Lane 6: 50µl PCR product, pSense66 (2300bp), 30-10-10
  7. Lane 7: BLANK
  8. Lane 8: 50µl PCR product, pSense66 (2300bp), colony (1/10µl H2O)


03102010-pcr2300bp.jpg