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
- 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.
- When the gel solidified, TAE buffer was added to the chamber until the gel was completely submerged in buffer.
- 10μL of DNA ladder pre-mixed with loading dye was loaded into the 1st well.
- 10μL of the M103S PCR product was mixed with 2μL of 6x loading buffer and loaded into the 2nd well.
- 10μL of the M103S control PCR product was mixed with 2μL of 6x loading buffer and loaded into the 3rd well.
- 10μL of the S33L PCR product was mixed with 2μL of 6x loading buffer and loaded into the 4th well.
- 10μL of the S33L control PCR product was mixed with 2μL of 6x loading buffer and loaded into the 5th well.
- The gel was run at 100V until the "dye line" moved about 3/4 of the way down the gel.
- The gel was placed in Ethidium Bromide Stain for about 30 minutes
- The gel was moved to TAE buffer to destain for about 20 minutes
- The gel was viewed and photographed under UV light.
- Be careful when working with ethidium bromide and UV light.
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.
- Mix 15μL of "cleaned-up" DNA with 5μL of 10x NEBuffer2, 25μL of sterile dH2O, and 5μL of HindIII enzyme.
- Incubate for two hours at 37°C.
- Incubate for 20 minutes at 65°C.
- Store at -20°C.
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.
- Place plastic culture tubes on ice for 15 minutes.
- Place DNA for transformation on ice.
- After DNA has thawed, place cells on ice. (Note - want to minimize the time that the cells are out of freezer and on ice).
- Add 1, 2, or 3uL of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute.
- 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.
- Put tube back in ice for 4 minutes.
- Heat shock at 42°C for 80 seconds.
- Add 100uL of SOC media.
- Shake at 37°C for 1 hour
- Plate 50uL of culture media.
- Incubate overnight at 37°C.