User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/12

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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 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).

Digestion of Original DNA in PCR Product

This was done with the 2nd round of PCR done yesterday for the S33L mutation.

  • Remove the wax from both tubes.
  • Add 1μL of DpnI to both PCR tubes.
  • Incubate the tubes on the heatblock at 37°C for one hour.
  • Remove and store at -20°C.

Making and Running a DNA Gel of Various PCR Products

  1. A 1.2% agarose gel was prepared by mixing 0.3g agarose with 25mL of TAE buffer. This mixture was microwaved for 37 seconds and then poured into a DNA gel chamber with a 15-well comb.
  2. When the gel solidified, TAE buffer was added to the chamber until the gel was completely submerged in buffer.
  3. 10μL of DNA ladder pre-mixed with loading dye was loaded into the 1st well.
  4. 10μL of the M103S PCR product was mixed with 2μL of 6x loading buffer and loaded into the 2nd well.
  5. 10μL of the M103S control PCR product was mixed with 2μL of 6x loading buffer and loaded into the 3rd well.
  6. 10μL of the S33L PCR product was mixed with 2μL of 6x loading buffer and loaded into the 4th well.
  7. 10μL of the S33L control PCR product was mixed with 2μL of 6x loading buffer and loaded into the 5th well.
  8. The gel was run at 100V until the "dye line" moved about 3/4 of the way down the gel.
  9. The gel was placed in Ethidium Bromide Stain for about 30 minutes
  10. The gel was moved to TAE buffer to destain for about 20 minutes
  11. The gel was viewed and photographed under UV light.
    • Be careful when working with ethidium bromide and UV light.

The Gel

Image:Photo_(14).JPG

Cutting with HindIII for Cloning

This was done with the DNA from the Gel Clean-Up earlier today. It was done for both the wildtype Asc Hb and the M8S/M33S/M103S Asc Hb clones.

  1. Mix 15μL of "cleaned-up" DNA with 5μL of 10x NEBuffer2, 25μL 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 at -20°C.

Transformations

Lots of transformations will be done today. The cells used will be either NovaBlue E.coli or M15MA E.coli. These were both made competent by me at different times. The procedure I will use today is a procedure from Dr. Hartings.

  • Plates were prepared by combining 12.5g of LB with 10g of Agar in 500mL of dH2O. This mixture was autoclaved on a liquid cycle. 500μL of 100mg/mL ampicillin was added to this when it cooled, and plates were then poured.


  1. Place plastic culture tubes on ice for 15 minutes.
  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 1, 2, or 3uL 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.
  11. Incubate overnight at 37°C.



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