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

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Making the M15MA 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.
  1. 100mL of LB in a 250mL Erlenmeyer flask was placed at 37°C at 150rpm for about 30 min to pre-warm.
  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 four hours.
    • The OD600 should have been 0.5, but I grew past this point to an OD600 of 1.06. This may affect the overall competency of the cells.
  4. The mixture was placed on ice for 5 minutes and then transferred to 50mL centrifuge tubes (divided evenly between two tubes).
  5. The tubes were spun at 4000g at 4°C for 5 minutes, and then for another 5 minutes. The supernatent was discarded.
  6. 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
  7. The resuspended pellets in buffer were then placed on ice for 110 minutes.
    • It only should have been 90 minutes, but the centrifuge was being used, so I kept the cells on ice until it was available.
  8. The cells were spun at 4000g at 4°C for 5 minutes.
  9. The supernatent was discarded and the pellet was resuspended in 4mL of TFB2.
    • 10 mM HEPES, 10 mM RbCl, 75 mM CaCl2, 15% glycerol
  10. The resuspended cells were alliquoted in 200μL amounts (with three 500μL amounts).
  11. The cells were placed at -80°C.

Making a Glycerol Stock

  • With some of the leftover cells from the 10mL LB overnight growth, a glycerol stock of M15MA was made:
    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.

Spectroscopic Studies of Hemoglobin

The triple mutant Asc Hb (M8S/M33S/M103S), which was purified and concentrated on 5/22/12, first had a spectrum taken of it.

  • A spectrum of 1mL of 25mM Tris, 50mM NaCl, pH 8, was taken as the baseline from 200-800nm.
  • Then, a spectrum of 15μL of the triple mutant (from Tube 1) and 985μL of 25mM Tris, 50mM NaCl, pH 8 was taken from 200-800nm.
  • The final concentration of this was determined to be 19.8μM. Calculations:
    • Tube 1: 24.2mg/mL÷18400Da = 0.0013M = 1.32mM = 1320μM
    • Dilution: (15μL÷1000μL) = 0.015
    • 1320μM × 0.015 = 19.8μM
  • The absorbance of the 25mM Tris, 50mM NaCl, pH 8 was subtracted from the protein absorbance values and the data was plotted:

Image: Triple_mutant_spectrum.JPG


  • A spectrum of 30μL of the triple mutant (from Tube 1) and 970μL of 25mM Tris, 50mM NaCl, pH 8 was taken from 200-800nm.
  • The final concentration of this spectrum was 39.6μM.
  • The absorbance of the 25mM Tris, 50mM NaCl, pH 8 was subtracted from the protein absorbance values and the data was plotted:

Image:Triple_mutant_spectrum_39.6.JPG



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