User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/05/17

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Making Glycerol Stocks

Note: only BL21(DE3)+M33S/M103S and BL21(DE3)+M8S/M33S/M103S colonies grew overnight

  • For each culture:
    1. Add 250μL of sterile 60% glycerol to a sterile 1 mL CryoTube vials
    2. Add 750μL of the overnight growth to the vial
    3. Mix well by pipetting up and down
    4. Place the vial in a -80°C freezer for long term storage

Expressing the Triple Mutant

  1. Centrifuge the cultures grown overnight at 3500rpm at 4°C for 15 minutes.
  2. Resuspend each of the pellets in 1mL of LB broth.
  3. Add each of the resuspended pellets with broth to one of the four 2800mL flasks made and autoclaved yesterday with 1L of sterile LB.
  4. Add 1mL 100mg/mL ampicillin to each flask.
    • 100mg/mL ampicillin prepared with 5mL of distilled water and 0.5g of ampicillin, mixed and sterile filtered.
  5. Add 1mL of 40mg/mL hemin in dimethylformamide to each of the flasks.
    • 40mg/mL hemin: 160mg hemin + 4mL dimethylformamide, mixed
  6. Incubate these flasks at 37°C at 165rpm for about two hours.
  7. Add 1mL of 0.1M IPTG to each flask. Continue to incubate at 37°C at 165rpm for about three more hours.
  8. Centrifuge these cells at 4500rpm at 4°C for 15 minutes.
  9. Resuspend the pellets in 50mM Tris 50mM NaCl, pH ~9 (sterile-filtered).
  10. Place resuspended cells in -20°C freezer.

Running a DNA Gel of Yesterday's PCR Product

  1. Make a 1.2% agarose gel (0.3g agarose + 25mL TAE buffer, microwave until boiling, then pour into gel chamber, and place comb for wells)
  2. When the gel has solidified, add TAE buffer to the chamber until the gel is completely submerged in buffer
  3. Load 10μL of DNA ladder into the 1st well
  4. Load 10μL of the experimental PCR product from yesterday with 2μL 6x loading buffer into the 3rd well (mix before pipetting into well)
  5. Load 10μL of the control PCR product from yesterday with 2μL 6x loading buffer into the 5th well (mix before pipetting into well)
  6. Run the gel at 100V until the "dye line" has moved about 3/4 of the way down the gel
  7. Place the gel in Ethidium Bromide Stain for about 30 minutes
  8. Move the gel to TAE buffer to destain for about 20 minutes
  9. View under a UV light
    • Be careful when working with ethidium bromide and UV light.

No DNA showed up on the gel in the PCR product lane (Lane 3). Will try to transform anyways.

Making Ampicillin Stocks

  1. Mix 1g of ampicillin with 10mL dH2O
  2. Sterile filter this solution
  3. Aliquot 1mL each into microcentrifuge tubes
  4. Label and store at -20°C for future use

Transformations

Transformations of the Asc Hb mutations M33S, M33S/M103S, and M8S/M33S/M103S into NovaBlue E.coli will be done in order to make glycerol stocks tomorrow. The wild-type plamids for Asc Hb and myoglobin (wt swMb pT7-7) will also be transformed into NovaBlue in order for glycerol stocks to be made. Also, the PCR product from yesterday (Asc Hb M8S/M33S/M103S/A71M) will be transformed into NovaBlue to be mini-prepped in the future. The following procedure will be followed for all of these transformations:

  1. Take a sterile microcentrifuge tube and place it on ice.
  2. Mix 50μL of NovaBlue Competent E.coli with 5μL of plasmid or PCR product in the cold, sterile tube.
  3. Incubate this mixture for 30 minutes on ice.
  4. Transfer the tube to a heat block at 42°C for 90 seconds, and then transfer the tube back to ice for 5 minutes.
  5. Add 200μL of SOC media to the cells/plasmid or PCR product.
  6. Shake the mixture at 165rpm at 37°C for one hour.
  7. Spread 100μL of the mixture on an LB plate with 100μg/mL ampicillin.
    • The LB plates were made with 5.25g LB, 4.2g Agar, and 210mL of distilled water. The mixture was autoclaved on a liquid cycle. When the mixture cooled to about 60°C 210μL of 100mg/mL ampicillin was added to it and it was poured approximately evenly into 6 sterile petri dishes.
  8. Incubate the plate overnight (inverted) at 37°C.



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