Gel Purification of Double-Digested pQE-80-L-Kan
- Add 5μL of 10% SDS solution (for a final concentration of 0.9% SDS) to the double digested pQE-80-L-Kan that was stored at -20°C over the weekend.
- This was done so the enzyme will not remain attached to the DNA during gel electrophoresis.
- A 1.2% agarose gel was loaded with 50μL of the double digested product with 10μL of 6x loading dye.
- 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 30 minutes.
- The gel was then destained in TAE buffer for about 20 minutes.
- After the band was cut out of the gel it was stored at 4°C for about an hour.
- The protocol for the Wizard® SV Gel and PCR Clean-Up System was then 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).
- Add approximately 5 colonies of BL21(DE3) + wt swMb pT7-7 to each flask with 50mL LB with 100μg/mL ampicillin.
- Incubate at 37°C at 200rpm all day and 250rpm O/N.
- Note: Growth was started at 11:18am.
- 100mL LB: 2.5g LB (w/o NaCl) + 1g NaCl + 100mL dH2O
- 50mL LB: 1.25g LB (w/o NaCl) + 0.5g NaCl + 50mL dH2O
All of these were autoclaved on a liquid cycle. dH2O was autoclaved as well.
- 1L terrific broth: 12g tryptone + 24g yeast extract + 4mL of glycerol, diluted to 900mL
- Four of these were made.
- Also, 4 100mL potassium phosphate solutions were made: 2.31g KH2PO4 + 12.54g K2HPO4
- The potassium phosphate solution will be added to the terrific broth before it is innoculated with cells.
All of these were autoclaved on a liquid cycle.
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.
- Mix 2μL of 10x T4 DNA Ligase Buffer, 4μL of the double-digested pQE-80-L-Kan vector, 12μL of the insert, 1μL of autoclaved dH2O, and 1μL of T4 DNA Ligase.
- Place at 16°C overnight.
- The thermocycler was used to maintain this temperature overnight.
Start Growth for Minipreps and Midipreps
All of these cultures were grown overnight at 37°C at 250rpm.