User:Meng Xiao He/Notebook/fall08/Constants

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