|
|
- For 50 ul reactions, prepare master mix as follows for n tubes:
- 10*n uL 5x Phusion buffer
- 4*n uL 2.5 mM dNTPs
- 2.5*n uL forward primer
- 2.5*n uL reverse primer
- 29.5*n uL PCR water
- Add 48 uL master mix to each of seven tubes.
- Label tubes blank, 0, 1, 2, 4, 8, 10
- Add DNA and MgCl2 to tubes according to chart:
|
| blank
| 0
| 1
| 2
| 4
| 8
| 10
|
| MgCl2 | 0 | 0 | .5 | 1 | 2 | 4 | 5
|
| DNA | 0 | 1 | 1 | 1 | 1 | 1 | 1
|
- Add 0.5 uL Phusion polymerase to each tube.
- Place tubes in thermocycler.
- Adjust settings as needed.
|
"
