Continuing to Make Wildtype Asc Hb "Apo"
- Move the dialysis tubing from the overnight dialysis solution to a fresh solution of 10mM NaHCO3.
- Prepared today: 1.68g sodium bicarbonate + 2L dH2O.
- Dialyze for about four hours in this solution with stirring.
- Then move the dialysis tubing from the 10mM NaHCO3 dialysis solution into 2L of dH2O.
- Dialyze for about four hours in the dH2O with stirring.
- Move the dialysis tubing from the dH2O into 2L more of dH2O.
- Dialyze overnight in the dH2O with stirring.
PCR for Cloning
This procedure was done for the wild-type Asc Hb (Hb pET 3d A) and the triple mutant Asc Hb (M8S/M33S/M103S):
- Mix 34.5μL of Nuclease-free water, 10μL of 5X Phusion HF Buffer, 1μL of 10mM dNTPs, 2.5μL forward primer, 2.5μL reverse primer, and 1μL of either plasmid.
- Add 0.5μL of Phusion DNA Polymerase, mix the solution, and centrifuge shortly.
- Put 50μL of wax on top of the mixture and place on the pre-heated block of the thermocycler.
- Start with 30 seconds at 98°C on the thermocycler.
- Cycle through the following 40 times:
- 10 seconds at 98°C
- 30 seconds at 62°C
- 15 seconds at 72°C
- A final extension step of 5 min was done at 72°C.
- The thermocycler was then held at 4°C.
Most components for this PCR came from the Phusion® High-Fidelity PCR Kit.
Running a DNA gel to Extract and Purify PCR Clones
- 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.
- When the gel had hardened the comb was removed and 10μL of DNA ladder was loaded into the first lane.
- 25μL of the wild-type PCR product with 5μL of 6x loading dye was loaded into the first wide lane.
- 25μL of the triple mutant PCR product with 5μL of 6x loading dye was loaded into the second wide lane.
- The gel was run at 90V until the dye band had moved about 3/4 of the way down the gel.
- The gel was then stained in ethidium bromide for about an hour.
Beginning the The Wizard® SV Gel and PCR Clean-Up System Protocol
The full protocol can be found here.
- The gel was viewed a photographed under UV light.
- Two 1.5mL microcentrifuge tubes were weighed and their weights were recorded.
- Tube for wild-type PCR: 1.05g
- Tube for triple mutant PCR: 1.03g
- The large bands on the gel (at ~0.5kb) were cut out of the gel with a new razor blade (additional safety required with UV light).
- The bands were placed into the microcentrifuge tubes and the tubes were re-weighed.
- Tube for wild-type PCR: 1.38g
- Tube for triple mutant PCR: 1.25g
- The microcentrifuge tubes will be stored at 4°C overnight.
Transformations are going to be done with the uncut pQE-80-L-Kan and double digested pQE-80-L-Kan, to make sure the pQE-80-L-Kan was cut previously on 06/15/12. The uncut plasmid (the midiprep product from 06/19/12 should transform, and the double-digested plasmid should not if it's actually cut.
- Place plastic culture tubes on ice for about 15 min.
- 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 1uL 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 on LB/amp plates (prepared 6/27/12: 6.25g LB (w/o NaCl) + 2.5g NaCl + 5g agar + 250mL dH2O, autoclave liquid cycle, left in the autoclave overnight, so to pour plates today I had to melt the LB/agar over a Bunsen burner flame, then I added 250μL of 100mg/mL ampicillin).
- Incubate inverted overnight at 37°C.
- For mini-prep: scrape NovaBlue + M8S/M33S/M103S glycerol stock with sterile wooden stick and inoculate 10mL of LB with 100μg/mL ampicillin, grow overnight at 37°C at 250rpm
- For making competent cells: scrape NovaBlue frozen competent cells (from -80°C freezer) with sterile wooden stick and inoculate 10mL of LB, grow overnight at 37°C at 250rpm