User:Meng Xiao He/Notebook/fall08/Constants

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(Platinum)
Current revision (12:31, 12 December 2008) (view source)
(Phusion)
 
Line 12: Line 12:
==Phusion==
==Phusion==
 +
PhFLANK:
 +
#2min at 98
 +
#7s at 98
 +
#30s @ 63->57 in 15 cycles (drop 0.4 degrees per cycle)
 +
#30s @ 59->56 in 15 cycles (drop 0.2 degrees per cycle)
 +
#30s @ 57 for 10 cycles
 +
#3:45 @ 72 (should capture WT cells as well). 45s for last 10
 +
#5 min@ 72 to end
 +
#hold at 4

Current revision

Contents

PCR

Platinum

cco MR-1 colony

12-7 on big room machine:

25ul reactions, 40 cycles:

  • 7 min initial 94C denaturation
  • 30 s denature
  • 30 s annealing 58→56.5 in first 15 cycles by dropping 0.1 decrease/cycle, 59→56.5 in last 25 cycles
  • 2 min elongation
  • 10 min final extension

Phusion

PhFLANK:

  1. 2min at 98
  2. 7s at 98
  3. 30s @ 63->57 in 15 cycles (drop 0.4 degrees per cycle)
  4. 30s @ 59->56 in 15 cycles (drop 0.2 degrees per cycle)
  5. 30s @ 57 for 10 cycles
  6. 3:45 @ 72 (should capture WT cells as well). 45s for last 10
  7. 5 min@ 72 to end
  8. hold at 4
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