Split PCR for M8S Mutation
Using a modified version of 2-stage QuikChange the M8S mutation should be inserted into the plasmid which already has the M33S and M103S mutations.
Protocol
First Stage
1st stage
| Tube |
sterile H2O |
Pfu Buffer |
M33S/M103S mutated hemoglobin |
For primer (M8S f) |
Rev primer (M8S r) |
dNTPs |
Pfu Turbo |
wax
|
|
|
(10X) |
(~100 ng/μL) |
(12.5 ng/μL) |
(12.5 ng/μL) |
(10 mM ea) |
(2.5 U/μL) |
|
| Experimental-forward
|
27 μL |
5 μL |
5 μL |
10 μL |
0 μL |
2 μL |
1 μL |
50 μL
|
| Experimental-reverse
|
27 μL |
5 μL |
5 μL |
0 μL |
10 μL |
2 μL |
1 μL |
50 μL
|
| (-) Control-forward
|
28 μL |
5 μL |
5 μL |
10 μL |
0 μL |
2 μL |
0 μL |
50 μL
|
| (-) Control-reverse
|
28 μL |
5 μL |
5 μL |
0 μL |
10 μL |
2 μL |
0 μL |
50 μL
|
- Combine all components of each mixture in sterile PCR tubes, except for Pfu Turbo and wax, mix well, but gently
- Add Pfu Turbo and mix gently with pipet
- Add wax on top of mixture and place on thermoclycler
- 30" @ 95°C
- Cycle through the following six times:
- 30" @ 95°C
- 1' @ 55°C
- 5' @ 68°C
- Remove from thermocycler, and pipet wax off of each reaction mixture
- Begin second stage
Second Stage
2nd stage
| Tube |
1st stage-forward |
1st stage-reverse |
Pfu Turbo |
wax
|
|
|
|
(2.5 U/μL) |
|
| Experimental
|
25 μL |
25 μL |
1 μL |
50 μL
|
| (-) Control
|
25 μL |
25 μL |
0 μL |
50 μL
|
- Combine mixtures from first stage and mix together gently with pipet, then add Pfu Turbo and mix again gently with pipet
- Add wax on top of mixture and place on thermoclycler
- 30" @ 95°C
- Cycle through the following 20 times:
- 30" @ 95°C
- 1' @ 55°C
- 5' @ 68°C
- 15' @ 68°C
- hold @ 4°C
Digestion of Original DNA
- Remove the wax from both tubes.
- Add 1μL of DpnI to both PCR tubes.
- Incubate the tubes on the heatblock at 37°C for one hour.
- Remove and store at -20°C
|
Project name
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