Thermocycle Conditions 7-10-13

2015-01-13

azim58 - 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

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