User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/04

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Continuing to Make Wildtype Asc Hb "Apo"

  1. Move the dialysis tubing from the overnight dialysis solution to a fresh solution of 10mM NaHCO3.
    • Prepared today: 1.68g sodium bicarbonate + 2L dH2O.
  2. Dialyze for about four hours in this solution with stirring.
  3. Then move the dialysis tubing from the 10mM NaHCO3 dialysis solution into 2L of dH2O.
  4. Dialyze for about four hours in the dH2O with stirring.
  5. Move the dialysis tubing from the dH2O into 2L more of dH2O.
  6. Dialyze overnight in the dH2O with stirring.

Cutting with HindIII for Cloning

This was done with the pQE-80-L-Kan midiprep from 06/19/12. Since it appeared that there were two smaller bands on the gel yesterday, I will try both cuts in NEBuffer4 instead of 2 because the BamHI has reduced star activity in this buffer, and maybe star activity could be affecting the cuts.

  1. For the pQE-80-L-Kan, mix 27μL of DNA with 5μL of 10x NEBuffer4, 13μL of sterile dH2O, and 5μL of HindIII enzyme.
  2. Incubate for two hours at 37°C.
  3. Incubate for 20 minutes at 65°C.
  4. Store on ice.

The Wizard® SV Gel and PCR Clean-Up System

The procedure from Promega was followed starting where I left off yesterday, but only for the wild-type double digest Asc Hb and triple mutant double digest Asc Hb. The DNA purification was done by centrifugation. The following deviations from the procedure were done:

  • The tabletop centrifuge we have only has a max speed of 13200rpm, so this speed was used instead of 14000rpm (which may affect yield).
  • The step to evaporate the additional ethanol with no cover on the centrifuge was done in a mini centrifuge for 1.5min (only has a max speed of about 6000rpm).

Making Media

250mL of SOB was made in a 2800mL flask by combining 5g of tryptone, 1.25g of yeast extract, 0.12g of NaCl, 625μL of 1M KCL, and 250mL of distilled H2O. Two of these were made and both were autoclaved on a liquid cycle. When cooled 1.25mL of 2M MgCl2 was added to each flask.

Starting Growth for Making Competent Cells

I will attempt to make competent cells with both the BL21(DE3) and NovaBlue strains of E.coli. 100μL of each strain was added to 250mL of SOB. The flasks were placed at 18°C at 250rpm to grow.

Cutting with BamHI for Cloning

  1. Mix 45μL of this morning's HindIII digest product with 5μL of BamHI-HF (HF=high fidelity).
  2. Incubate for two hours at 37°C.
  3. Add 5μL of 10% SDS solution (for a final concentration of 0.91% SDS).
    • This was done so the enzyme will not remain attached to the DNA during gel electrophoresis.

Running an Analytical/Gel Purification DNA Gel of the pQE-80-L-Kan HindIII/BamHI Product from This Morning

  1. A 1.2% agarose gel was made by mixing 0.3g of agarose with 25mL TAE buffer. This mixture was heated in the microwave for 37 seconds and then poured into a gel chamber with a comb inserted.
  2. When the gel had hardened the comb was removed and 5μL of pre-mixed DNA ladder was loaded into the first lane.
  3. 5μL of the midiprepped pQE-80-L-Kan with 1μL of 6x loading dye was loaded into the second lane.
  4. 50μL of the double digested pQE-80-L-Kan with 10μL of 6x loading dye was loaded into the first wide lane.
  5. The gel was run at 100V until the dye band had moved about 3/4 of the way down the gel.
  6. The gel was then stained in ethidium bromide for about 30 minutes.

Image:Photo_(27).JPG

The bands are very light (too much sunshine coming in the window), but the double digest pQE-80-L-Kan can be seen in the wide lane as being the correct size this time.

Gel Purification of Double-Digested pQE-80-L-Kan

  1. The protocol for the Wizard® SV Gel and PCR Clean-Up System was followed: procedure. The DNA purification was done by centrifugation. The following deviations from the procedure were done:
    • The tabletop centrifuge we have only has a max speed of 13200rpm, so this speed was used instead of 14000rpm (which may affect yield).
    • The step to evaporate the additional ethanol with no cover on the centrifuge was done in a mini centrifuge for 1.5min (only has a max speed of about 6000rpm).

DNA ligation with T4 DNA Ligase

This will be done with both the wildtype Asc Hb double-digested insert and the M8S/M33S/M103S Asc Hb double-digested insert. The procedure is based off of the protocol on the NEB website.

  1. Mix 2μL of 10x T4 DNA Ligase Buffer, 7μL of the double-digested pQE-80-L-Kan vector, 10μL of the insert, and 1μL of T4 DNA Ligase.
  2. Place at 16°C overnight.
    • The thermocycler was used to maintain this temperature overnight.



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