User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/05

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Reconstitute Asc Hb with Nickel

Instead of doing two separate reconstitutions like planned, I combined the two dialysis tubes for more apoprotein in the reaction for hopefully a higher yield. The following procedure took place in a cold room (4°C):

  1. The apoprotein from the dialysis tubing was poured into a beaker and placed on a stir plate with a stir bar.
  2. 4mL of 1M Potassium Phosphate buffer (pH 7.1) was added dropwise (very slowly) to the protein (while gently stirring) over a 20 minute period.
  3. Ni(protoporphyrin IX)solution was added dropwise (pretty slowly) to the apoprotein (while gently stirring).
    • The Ni(protoporphyrin IX)solution was prepared by dissolving 2mg of Ni(protoporphyrin IX) in 10 drops of 0.1N NaOH, and then adding 4.5mL of dH2O. 2mL of 0.1M Potassium Phosphate buffer (pH 7.1) was added to this solution. The solution was mixed on a stir plate.
  4. The beaker with the apoprotein/Ni(protoporphyrin IX) mixture was wrapped in aluminum foil and left to gently stir in the cold room.
  5. 12 hours later Ni(protoporphyrin IX)solution was added again dropwise (pretty slowly) to the apoprotein (while gently stirring).
    • The Ni(protoporphyrin IX)solution was prepared by dissolving 2mg of Ni(protoporphyrin IX) in 10 drops of 0.1N NaOH, and then adding 4.5mL of dH2O. 2mL of 0.1M Potassium Phosphate buffer (pH 7.1) was added to this solution. The solution was mixed on a stir plate.
  6. The beaker with the apoprotein/Ni(protoporphyrin IX) mixture was wrapped in aluminum foil and left to gently stir in the cold room for another 12 hours.

Running an Analytical DNA Gel

  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 5μL of DNA ladder into the 2nd well.
  4. Load 5μL of the wild-type Asc Hb and pQE-80-L-Kan ligation from overnight with 1μL 6x loading buffer into the 4th well (mix before pipetting into well).
  5. Load 5μL of the triple mutant (M8S/M33S/M103S) Asc Hb and pQE-80-L-Kan ligation from overnight with 1μL 6x loading buffer into the 5th well (mix before pipetting into well).
  6. Load 5μL of the midiprepped pQE-80-L-Kan plasmid from 06/19/12 with 1μL 6x loading buffer into the 6th well (mix before pipetting into well).
  7. Run the gel at 100V until the "dye line" has moved about 3/4 of the way down the gel.
  8. Place the gel in Ethidium Bromide Stain for about 30 minutes.
  9. View under a UV light.
    • Be careful when working with ethidium bromide and UV light.

Image:Photo_(28).JPG

There is no proof the ligation worked on this gel (maybe the concentration of DNA is too low to see). Transformations will be done today to see if there is a ligation product to transform.

Making Buffer for Making Competent Cells

The buffer will be referred to as TB.

  1. Combine 0.48g HEPES, 3.73g KCl, and 0.33g CaCl2 in a beaker.
  2. Add about 150mL of dH2O and stir on a stir plate.
  3. pH the buffer to 6.7 with 1M KOH.
  4. Add 2.18g MnCl2 to the buffer.
  5. Dilute the buffer to 200mL with dH2O.
  6. Filter.

Transformations of Ligation Products

This procedure was done for both ligation products from the overnight ligation (wild-type Asc Hb + pQE-80-L-Kan ligation and triple mutant (M8S/M33S/M103S) Asc Hb + pQE-80-L-Kan ligation):

  1. Place plastic culture tubes on ice for about an hour.
  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 1uL of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute.
  5. Add cells (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 100uL of culture media on LB/amp plates (prepared fresh today: 6.25g LB (w/o NaCl) + 2.5g NaCl + 5g agar + 250mL dH2O, autoclave liquid cycle, cool to about 60°C, add 250μL 100mg/mL amicillin, pour plate).
  11. Incubate inverted overnight at 37°C.

Making Cells Competent

This procedure was done with both the NovaBlue and BL21(DE3) E.coli.

  1. The NovaBlue cells were grown to an OD600 of about 0.8 and the BL21(DE3) E.coli grew to an OD600 of about 0.56 (the target was 0.6).
  2. The cells were incubated on ice for 10 minutes.
  3. The cells were then spun at 2500g for 10 minutes at 4°C.
  4. The pellets were each resuspended in 80mL of ice-cold TB.
  5. The cells were then spun again at 2500g for 10 minutes at 4°C.
  6. # The pellets were each resuspended in 20mL of ice-cold TB.
  7. 1.5mL of DMSO was added slowly with gentle swirling to the cells (final concentration of about 7%).
  8. The cells were alliquotted, frozen in liquid nitrogen, and stored at -80°C.



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