Thermocycle+Conditions+7-9-13

Thermocycle Conditions 7-9-13 has been associated with pm ss tdwn (pm = primestar max, ss = small primer start, tdwn = touchdown)

95 C 15 s, 95 C hold, 95 C 2 min, 95 C hold, 98 C 10 s, 65 C 10 s, 72 C 60 s, 98 C 10 s, 64 C 10 s, 72 C 60 s,  98 C 10 s, 63 C 10 s, 72 C 60 s,  98 C 10 s, 62 C 10 s, 72 C 60 s,  98 C 10 s, 61 C 10 s, 72 C 60 s,  98 C 10 s, 60 C 10 s, 72 C 60 s,  98 C 10 s, 59 C 10 s, 72 C 60 s,  98 C 10 s, 58 C 10 s, 72 C 60 s,  98 C 10 s, 57 C 10 s, 72 C 60 s,  98 C 10 s, 56 C 10 s, 72 C 60 s,  (98 C 10 s, 55 C 10 s, 72 C 60 s)X15 4 C hold, 98 C hold (98 C 10 s, 55 C 10 s, 72 C 60 s)X20 4 C hold (98 C 10 s, 55 C 10 s, 72 C 60 s)X15 4 C hold (98 C 10 s, 55 C 10 s, 72 C 60 s)X10 4 C hold

1st part to warm up thermocycler 2nd part to denature 3rd part to add polymerase and denature further 10 decreasing annealing temperature cycles for touchdown amplification for 15 cycles with the small amount of primer originally added (1 uL of 2 uM forward and reverse primer each; 0.04 uM final concentration each primer at this point) 98 C hold for addition of more primer (1 uL of 10 uM forward and reverse primer each; 0.24 uM final concentration each primer at this point) 20 cycles of amplification 4 C hold for removal of an aliquot if desired (e.g. 20 uL) 15 cycles of amplification 4 C hold for removal of an aliquot if desired (e.g. 20 uL) 10 cycles of amplification 4 C hold for removal of remaining sample (e.g. 10 uL)

Timing -time from addition of polymerase to addition of more primer: 80s*10+80s*15 = 2000s = 33.33m -time from addition of more primer to removal of 1st aliquot: 80s*20 = 1600s = 26.67m -time from removal of 1st aliquot to removal of 2nd aliquot: 80s*15 = 1200s = 20m -time from removal of 2nd aliquot to removal of final aliquot: 80s*10 = 800s = 13.33m

see also PCR by starting with a small amount of primer and later adding more primer and polymerase portal 4-11-13