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
 * 3) Tube 1: pUA66katE 25-10-10-BLK
 * 4) Tube 2: pUA66katE 25-10-10-NA
 * 5) Tube 3: pUA66katE 30-10-10
 * 6) 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)




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