User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/14

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Results from Overnight Growths

  • The transformation of wt swMb pT7-7 (myoglobin) into BL21(DE3) worked perfectly (lots of colonies). This can be expressed next week.
  • The transformation of the M8S/M33S/M103S/A71M Asc Hb PCR product into NovaBlue cells got a few colonies with some of the 40μL amounts of cells. Three of these colonies will be grown in LB/amp overnight tonight to make sure they are actually transformed, and if they grow, the cultures will be mini-prepped.
  • The test to see if the transformations from two nights ago worked showed that the M15MA cells really were transformed with the test plasmid (grew in LB/amp overnight), but also showed that none of the NovaBlue cells were really transformed.

Continuing Expression of Wildtype Hemoglobin

  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. Incubate these flasks at 37°C at 165rpm until OD600=1.2.
  6. Add 1mL mixture of 0.1M IPTG, 1M FeCl3, 0.21M alpha aminolevulinic acid to each flask. Continue to incubate at 37°C at 165rpm overnight.
    • Mixture prepared: 1.5g FeCl3 in 9mL dH2O, let cool. Add 0.22g IPTG and 0.25 alpha aminolevulinic acid to the solution.
    • Two of the flasks had to be removed from the shaker for an hour while the transformation tubes were in there.

The protein made in this expression was purified by Dr. Hartings. Details for the next steps can be found here.

Making BL21(DE3) Cells Competent

  • The following procedure will be used to make competent cells: [[1]]. The only change will be that HEPES will be used instead of MOPS. The buffer will still be made to the same pH.
  • I forgot to prepare the 100mL of LB yesterday, so there was a delay in starting the procedure this morning. The 10mL culture that was grown overnight was placed on ice while the LB was autoclaved.
  1. 100mL of LB in a 250mL Erlenmeyer flask was placed at room temperature until it cooled to about 37°C.
  2. 1mL of the overnight culture was transferred to the pre-warmed flask and was incubated at 37°C at 250rpm.
  3. The mixture was grown for two hours.
  • The OD600 at this point was 0.49.
  1. The mixture was placed on ice for 5 minutes and then transferred to 50mL centrifuge tubes (divided evenly between two tubes).
  2. The tubes were spun at 4000g at 4°C for 5 minutes, and then for another 5 minutes. The supernatent was discarded.
  3. The pellets were resuspended in a total volume of 30mL of the TFB1.
    • 100 mM RbCl, 50 mM MnCl2, 30 mM Potassium acetate, 10 mM CaCl2, 15% glycerol, this solution was sterile-filtered today
  4. The resuspended pellets in buffer were then placed on ice for 90 minutes.
  5. The cells were spun at 4000g at 4°C for 5 minutes.
  6. The supernatent was discarded and the pellet was resuspended in 4mL of TFB2.
    • 10 mM HEPES, 10 mM RbCl, 75 mM CaCl2, 15% glycerol
  7. The resuspended cells were alliquoted in 200μL amounts (with three 500μL amounts).
  8. The cells were placed at -80°C.

Preparing the Qiagen Vector for Cloning

First I needed to prepare TE buffer.

  1. 0.5M EDTA was made by dissolving 18.6g EDTA in dH2O. The pH was increased to 8 and small amounts of heat were applied so that it would dissolve. The final volume was 100mL.
  2. The TE buffer was made by mixing 1mL of 1M Tris (pH 7.5) with 0.2mL 0.5M EDTA, and dH2O to a final volume of 100mL.
  3. 15mL of this buffer was sterile-filtered.

The vector that was sent to us, which came as 25μg, was diluted to 0.5μg/μL by adding 50μL of the sterile-filtered TE buffer. This was stored at -20°C.

Transformations

Transformations were done with the M8S/S33L/M103S Asc Hb into NovaBlue cells (lots of different amounts of cells and DNA). One transformation was also done with pQE-80-L-Kan into NovaBlue cells (1μL DNA, 50μL cells).

  • Plates were prepared by combining 6.25g of LB (w/o NaCl) with 2.5g of NaCl and 5g of Agar in 250mL of dH2O. This mixture was autoclaved on a liquid cycle. 250μL of 100mg/mL ampicillin was added to this when it cooled, and plates were then poured.


  1. Place plastic culture tubes on ice for 15 minutes.
  2. Place DNA for transformation on ice.
  3. After DNA has thawed, place cells on ice. (Note - want to minimize the time that the cells are out of freezer and on ice).
  4. Add 1, 2, or 3uL of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute.
  5. Add cells (30uL, 40uL, or 50uL) to the tube on top of the DNA and gently pipette up and down to mix cells and DNA.
  6. Put tube back in ice for 4 minutes.
  7. Heat shock at 42°C for 80 seconds.
  8. Add 100uL of SOC media.
  9. Shake at 37°C for 1 hour.
  10. Plate 50uL of culture media.
  11. Incubate overnight at 37°C.


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