User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/12

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Reconstitute Asc Hb with Manganese

  1. Mn-porphyrin solution was again added dropwise (pretty slowly) to the apoprotein (while gently stirring).
    • The Mn-porphyrin solution was prepared by dissolving 2mg of Mn-porphyrin 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.
  2. The beaker with the apoprotein/Mn-porphyrin mixture was wrapped in aluminum foil and left to gently stir in the cold room overnight.

Cutting with HindIII for Cloning

This was done with the PCR clones (wt Asc Hb and M8S/M33S/M103S Asc Hb) that were gel-purified yesterday and the pQE-80-L-Kan midiprep from 06/19/12. This will be done in NEBuffer4 instead of 2 because the BamHI has reduced star activity in this buffer, and maybe star activity could be affecting the cuts that lead to poor ligations.

  1. For the each DNA sample, mix 40μL of DNA with 5μL of 10x NEBuffer4, and 5μL of HindIII enzyme.
  2. Incubate for two hours at 37°C.
  3. Incubate for 20 minutes at 65°C.

Making Cells Competent

The NovaBlue cells this morning were at an OD600 of 1.5 (overgrown). The BL21(DE3) cells were only at 0.2 so they will continue to grow at 18°C. For the NovaBlue cells:

  1. The cells are the put on ice for 10-15 minutes and then spun at 4500rpm for 15 minutes.
  2. The pellet is resuspended in 83.3mL of TFB.
    • TFB is standard transformation buffer and is 10mM K-MES, 100mM RbCl, 45mM MnCl2, 10mM CaCl2, and 3mM HACoCl3, with 10% glycerol. This buffer was prepared and sterile filtered in November 2011 (before OpenWetWare was cool).
  3. The resuspended pellet is put on ice for 10 minutes and then spun at 4500rpm for 15 minutes.
  4. The pellet of this is then resuspended in 20mL of TFB.
  5. Then, 700µl of DMSO is added, then 700µl of 2.25M DTT is added, and finally another 700µl of DMSO is added.
  6. The mixture is put on ice for 5 minutes and then alliquoted into sterile microcentrifuge tubes.
  7. The tubes are frozen with liquid nitrogen and stored long-term at -80°C.
  • This procedure is based on the paper “Studies on Transformation of Escherichia Coli with Plasmids” by Douglas Hanahan.

Making Wildtype Asc Hb "Apo"

This procedure was done twice, with two 20mL portions of the wildtype Asc Hb:

  1. In the cold room 20mL of 2-butanone was added to the wildtype Asc Hb in 25mM Tris (pH 8) protein and the protein was shaken vigorously for 30 seconds.
  2. Out of forgetfulness I forgot to lower the pH of the protein. I realized this when a separation wasn't seen after waiting a few minutes. 230μL of 1M HCl was added to 20mL of protein plus 20mL of 2-butanone. The mixture was re-shaken.
    • This step hopefully lowered the pH of the protein to 2.3
  3. The solution sat for about five minutes and then a clear seperation was seen (the top half was pink and the bottom half was almost clear). The top half (the heme in 2-butanone) was removed by a Pasteur pipette.
  4. 20mL more of 2-butanone was added to the protein, shaken vigorously for 30 seconds, allowed to sit for five minutes, and then when separation was seen the top half was removed by a Pasteur pipette.
  5. The previous step was repeated one more time.
  6. The protein layer was then transferred to dialysis tubing.
  7. The dialysis tubing was placed in 2L of 10mM NaHCO3.
    • Prepared today: 1.68g sodium bicarbonate + 2L dH2O.
  8. The protein was dialyzed for four hours (with stirring).
  9. The dialysis tubing was moved to a fresh 2L of 10mM NaHCO3.
    • Prepared today: 1.68g sodium bicarbonate + 2L dH2O.
  10. The protein was dialyzed overnight (with stirring).

Cutting with BamHI for Cloning

  1. Mix 45μL of this morning's HindIII digest products with 5μL of BamHI-HF (HF=high fidelity).
  2. Incubate for two hours at 37°C.
  • I forgot to add SDS before electrophoresis. Hopefully, this error will not affect my DNA.

Running a DNA Gel of Today's Double Digest Products

  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 1st well.
  4. Load 50μL of the double-digested wildtype Asc Hb from today into the first wide well.
  5. Skip the second wide well (because it's broken).
  6. Load 50μL of the double-digested M8S/M33S/M103S Asc Hb from today into the third wide 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_(32).JPG

It turns out that I stained the gel in the container that is used as a destain. Since the destain hasn't been changed in a while there is some ethidium bromide present, which is why the gel did stain, but that might explain why the bands are not very bright.

Transformations

Transformations were done with the NovaBlue cells that were made competent today. The pQE-80-L-Kan midiprep product from 06/19/12 was used as a positive control to make sure the cells were actually made competent. The ligation products from last night (pQE-80-L-Kan with either wildtype Asc Hb or M8S/M33S/M103S Asc Hb).

  1. Place plastic culture tubes on ice for 15 minutes.
  2. Place DNA for transformation on ice.
  3. Add 5L of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute.
  4. Add 50uL of cells to the tube on top of the DNA and gently pipette up and down to mix cells and DNA.
    • This portion of cells was not frozen with the rest of the cells this morning, it was just kept on ice until used.
  5. Put tube back in ice for 5 minutes.
  6. Heat shock at 42°C for 30 seconds.
  7. Add 100uL of SOC media.
  8. Shake at 250rpm at 37°C for 1 hour
  9. Plate 50uL of culture media on an LB plate with 100μg/mL ampiciliin .
  10. Incubate overnight at 37°C.

Making More Cells Competent

Later on in the day the BL21(DE3) cells had grown to an OD600 of 0.7. At this point, the following procedure was followed:

  1. The cells are the put on ice for 10-15 minutes and then spun at 4500rpm for 15 minutes.
  2. The pellet is resuspended in 83.3mL of TFB.
    • TFB is standard transformation buffer and is 10mM K-MES, 100mM RbCl, 45mM MnCl2, 10mM CaCl2, and 3mM HACoCl3, with 10% glycerol. This buffer was prepared and sterile filtered in November 2011 (before OpenWetWare was cool).
  3. The resuspended pellet is put on ice for 10 minutes and then spun at 4500rpm for 15 minutes.
  4. The pellet of this is then resuspended in 20mL of TFB.
  5. Then, 700µl of DMSO is added, then 700µl of 2.25M DTT is added, and finally another 700µl of DMSO is added.
  6. The mixture is put on ice for 5 minutes and then alliquoted into sterile microcentrifuge tubes.
  7. The tubes are frozen with liquid nitrogen and stored long-term at -80°C.
  • This procedure is based on the paper “Studies on Transformation of Escherichia Coli with Plasmids” by Douglas Hanahan.

Beginning the The Wizard® SV Gel and PCR Clean-Up System Protocol

This was done for the two double digest products that were run on a gel today. The full protocol can be found here.

  1. Two 1.5mL microcentrifuge tubes were weighed and their weights were recorded.
    • Tube for wild-type double digest: 1.03g
    • Tube for triple mutant double digest: 1.04g
  2. The large bands on the gel were cut out of the gel with a razor blade (additional safety required with UV light).
  3. The bands were placed into the microcentrifuge tubes and the tubes were re-weighed.
    • Tube for wild-type double digest: 1.23g
    • Tube for triple mutant double digest: 1.22g
  4. The microcentrifuge tubes will be stored at 4°C overnight.



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