User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/05/16

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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:
      1. 30" @ 95°C
      2. 1' @ 55°C
      3. 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:
      1. 30" @ 95°C
      2. 1' @ 55°C
      3. 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:

  1. Mix 3mL LB with 3μL of 100mg/mL ampicillin in a sterile test tube.
  2. Using a sterile, wooden stick, touch a colony on a plate and then swirl the stick in the LB/Amp.
  3. Incubate these mixtures overnight at 37°C at 250rpm.

Beginning of Expression

  1. Add 25μL of 100mg/mL ampicillin to 25mL LB.
  2. Using a sterile, wooden stick, touch a colony on a plate and then swirl the stick in the LB/Amp.
  3. Repeat for three other LB cultures.
  4. Incubate these mixtures overnight at 37°C at 250rpm.



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