Diluting PCR Primers
- A71M reverse primer:
- currently 27.8 nMoles or 0.36mg
- Add 278μL sterile dH2O to this to make a 100μM stock
- Take 5μL of 100μM primer and mix with 513μL sterile dH2O for 12.5ng/μL stock
- A71M forward primer:
- currently 27.4 nMoles or 0.35mg
- Add 274μL sterile dH2O to this to make a 100μM stock
- Take 5μL of 100μM primer and mix with 506μL sterile dH2O for 12.5ng/μL stock
Split PCR for A71M Mutation
Using a modified version of 2-stage QuikChange the A71M mutation should be inserted into the plasmid which already has the M8S, M33S, and M103S mutations.
Protocol
First Stage
1st stage
Tube |
sterile H2O |
Pfu Buffer |
M8S/M33S/M103S mutated hemoglobin |
For primer (A71M f) |
Rev primer (A71M 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 Wild-type DNA
- 1μL of DpnI was added to the PCR product and the control tube and they were put on a heat block at 37°C for one hour.
Making Media and Other things for Expression
- Make 4 1L LB broths: each is 25g LB with 1L of dH2O
- Make 4 25mL LB broths: each is 0.63g with 25mL of dH2O
- Autoclave all of these broths
- Prepare 5mL 0.1 M Isopropyl β-D-1-thiogalactopyranoside (IPTG) solution
- 0.12g + 5mL distilled H2O
- Sterile Filter the solution
- Prepare 5mL 100mg/mL Ampicillin
- 0.5g ampicillin + 5mL distilled H2O
- Sterile Filter the solution
Making More Things to Autoclave
- 2 50mL LB broths: 1.25g LB + 50mL dH2O
- 60% glycerol: 30mL glycerol + 20mL dH2O
- Media for LB plate: 0.875g LB + 0.7g agar + 35mL dH2O
- All of the above were autoclaved
- Also, autoclaved two dry cycles of centrifuge tubes, pipet tips, microcentrifuge tubes, etc.
Attempt to Make Glycerol Stocks
Some of these plates are pretty old, but I'm going to see if I can get colonies from plates that look uncontaminated to grow in LB and ampicillin overnight:
- Mix 3mL LB with 3μL of 100mg/mL ampicillin in a sterile test tube.
- Using a sterile, wooden stick, touch a colony on a plate and then swirl the stick in the LB/Amp.
- Incubate these mixtures overnight at 37°C at 250rpm.
Beginning of Expression
- Add 25μL of 100mg/mL ampicillin to 25mL LB.
- Using a sterile, wooden stick, touch a colony on a plate and then swirl the stick in the LB/Amp.
- Repeat for three other LB cultures.
- Incubate these mixtures overnight at 37°C at 250rpm.
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