User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/26

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The Wizard® SV Gel and PCR Clean-Up System

The procedure from Promega was followed starting where I left off yesterday. The pQE-80-L-Kan sample that was digested yesterday will not be "cleaned-up" today because it did not appear to be the correct size on the DNA gel from yesterday. 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 Glycerol Stocks

Glycerol stocks were made of 3 colonies of NovaBlue + M103S and 3 colonies of NovaBlue + M8S/M33L/M103S (the cultures that grew overnight for the miniprep).

  • For each culture:
    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

Miniprep of M8S/M33S/M103S/A71M Asc Hb

Minipreps were done for 3 colonies of NovaBlue + M103S and 3 colonies of NovaBlue + M8S/M33L/M103S (all colonies grew overnight). The vacuum protocol was followed: [1]

Quantifying DNA

The absorbances of the double-digested wildtype Asc Hb and and double-digested M8S/M33S/M103S Asc Hb ("that were cleaned-up") this morning) were taken at 260 ans 280nm in oreder to calculate the DNA concentration. The following procedure was repeated for both samples:

  1. Using a smaller volume quartz cuvette, 95μL of dH2O was placed in the spectrophotometer and the baseline was corrected with this.
  2. 5μL of DNA was added to the 95μL of water. This was pipetted up and down with a pipette to mix.
  3. The absorbance was taken at 260 and 280nm.

Results

Note: the absorbances are for 20x dilute DNA samples, and the absorbances may be low, but there is only a small volume of DNA to work with, so I don't want to use it all for this (so these concentrations may be off)

double-digested wildtype Asc Hb

  • A260 = 0.022
  • A280 = 0.018
  • Absorbance Ratio = 1.2402
  • 20x Dilute DNA concentration = 1.1100 μg/mL
  • Non-dilute DNA concentration = 22.2 μg/mL

double-digested M8S/M33S/M103S Asc Hb

  • A260 = 0.007
  • A280 = 0.004
  • Absorbance Ratio = 1.7805
  • 20x Dilute DNA concentration = 0.3650 μg/mL
  • Non-dilute DNA concentration = 7.3 μg/mL

DNA ligation with T4 DNA Ligase at Room Temperature

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, 2μL of the double-digested pQE-80-L-Kan vector, 15μL of the wildtype insert, and 1μL of T4 DNA Ligase.
  2. Mix 2μL of 10x T4 DNA Ligase Buffer, 2μL of the double-digested pQE-80-L-Kan vector, 15μL of the triple mutant insert, and 1μL of T4 DNA Ligase.
  3. Place at room temperature for 10 minutes.
  4. Place on ice.

Transformations

Transformations were done with the ligation products made today into NovaBlue cells.

  1. Place plastic culture tubes on ice for about 15 min.
  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 5uL 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 on LB/amp plates (prepared 6/21/12: 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, store at 4°C).
  11. Incubate inverted overnight at 37°C.

DNA ligation with T4 DNA Ligase at 16°C

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, 2μL of the double-digested pQE-80-L-Kan vector, 15μL of the wildtype insert, and 1μL of T4 DNA Ligase.
  2. Mix 2μL of 10x T4 DNA Ligase Buffer, 2μL of the double-digested pQE-80-L-Kan vector, 15μL of the triple mutant insert, and 1μL of T4 DNA Ligase.
  3. Place at 16°C overnight (using the thermocycler to maintain temperature).