Thermocycle+Conditions+7-10-13

Thermocycle Conditions 7-10-13 has been associated with pm tdwn (pm = primestar max, tdwn = touchdown)

95 C 15 s, 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)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)X15 4 C hold

1st part to warm up thermocycler 10 decreasing annealing temperature cycles for touchdown 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) 15 cycles of amplification 4 C hold for removal of remaining sample (e.g. 10 uL)

Timing -time to removal of 1st aliquot: 80s*30 = 2400s = 40m -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*15 = 1200s = 20m

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