User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/05/22

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Test Plasmid Transformation Results

The test plasmid transformation did not have any colonies when I arrived this morning. I decided to leave the plate at 37°C for the day to see if the cells were just slow-growing. No colonies were visible at the end of the day.

Split PCR for A71M Mutation

The PCR to introduce the A71M mutation last week did not have a band on the DNA gel that was run, and there have been transformation problems lately. Because of these problems, I have decided to repeat the PCR procedure from last week.

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 29 μL 5 μL 2 μL 10 μL 0 μL 2 μL 1 μL 50 μL
Experimental-reverse 29 μL 5 μL 2 μL 0 μL 10 μL 2 μL 1 μL 50 μL
(-) Control-forward 30 μL 5 μL 2 μL 10 μL 0 μL 2 μL 0 μL 50 μL
(-) Control-reverse 30 μL 5 μL 2 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 50mL LB broths: each is 1.25g with 50mL of dH2O
    • Autoclave all of these broths
  • Prepare 5mL 100mg/mL Ampicillin
    • 0.5g ampicillin + 5mL distilled H2O
    • Sterile Filter the solution

Triple Mutant Purification

A 5mL anion-exchange column was used for this purification.

  1. The column and pumps were prepared for the purification using water and buffer.
  2. 25mL of 25mM Tris, 50mM NaCl, pH 7.99 was run through the column at 5mL/min.
  3. 30mL of filtered supernatent (containing the protein with the triple mutant) in buffer was loaded onto the column at 5mL/min.
  4. 25mL of 25mM Tris, 50mM NaCl, pH 7.99 was run over the column at 5mL/min to wash away other proteins.
  5. A gradient of buffer, pH 8, with 25mM Tris, was run over the column over a half hour period (at 5mL/min) from 50mM NaCl to 0.5M NaCl, to elute off the protein. This flowthrough was collected in 5mL fractions.
  6. 25mL of 25mM Tris, 0.5M NaCl, pH 8 was run over the column to ensure it was clean.
  7. The procedure was then repeated from step 2, except this time 50mL of filtered supernatent in buffer was loaded onto the column.
  • The absorbance of what was coming off the column was monitored at 410nm during the purification to see where the protein was. The fractions that had some absorbance at this wavelength were pooled together, and concentrated in a Jumbosep™ Centrifugal Device. The protein was stored at 4°C.

Expressions

Expressions will be done using a new procedure with the wildtype Asc Hb and the triple mutant (M8S/M33S/M103S) Asc Hb.

  1. Add 50μL of 100mg/mL ampicillin to 50mL LB.
  2. Using a sterile, wooden stick, scrape the glycerol stock and then swirl the stick in the LB/Amp.
  3. Repeat for three other LB cultures. Two with wild-type and two with the triple mutant.
  4. Incubate these mixtures overnight at 37°C at 250rpm.


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