Plasmid digest (Dpn1 digest)

• Set-up the reaction mixture:

  Restriction Enzyme	1µl
  DNA	1 µg
  10X Cutsmart	5 µl (1X)
  Total Reaction Volume	50 µl

• Incubate for 1 hr at 37C
• Heat inactivate at 80C for 20 minutes

Heat shock transformation

• Incubate 50ng of plasmid construct with 25 µL of chemically competent E. coli T7
• Express cells or E. coli DH5α on ice for 30 minutes.
• Heat shock the cells for 45 seconds at 42C and placed back onto ice for 2 minutes.
• Allow cells to grow for 45 minutes in 200 µL of SOC outgrowth media (NEB) at 37°C and 200 rpm.
• Spread plate onto Luria broth (LB) agar plates containing 100 µg/mL of ampicillin and grown at 37°C overnight.

Colony PCR

Sequencing

• Transfer 10 uL of purified plasmid sample (50-100ng/uL) to an Eppendorf tube. Add 5 uL of one primer.
• Request sequencing. http://sequencing.ramaciotti.unsw.edu.au/index.html
• Label Eppendorf tubes with the order number, and 1, 2, 3, etc.
• Take Eppendorf tubes to the new Biosciences building Lvl 2 (at UNSW, Sydney, Australia), and store the samples in the fridge provided.
• Sanger sequencing is carried out following the provided protocol.

Miniprep

• Protocols were followed from the Qiagen QIAprep Spin Miniprep Kit. No changes were made.

Starter culture

• One colony was selected from the plate grown overnight and grown in 2 mL of LB containing 2 uL of the appropriate antibiotic at 37°C and 200 rpm and left overnight.

Large-scale grow-up

• Baffled shake flasks containing 500 mL of LB with 50uL of the appropriate antibiotic at 37°C are inoculated with the starter culture.
• The cells are grown at 37°C and 200 rpm and OD600 is periodically measured.
• Once OD600 reaches above 0.6, add IPTG of 1 mM concentration to induce the expression the proteins.
• After induction, grow the cells overnight at 24°C, 200 rpm.

Collection of cells by centrifugation

• Centrifuge the bacterial culture at 4600 x g for 20 minutes.
• Collect cell pellet and resuspended in binding buffer (20mm NaH2PO4, 500mM NaCl, 10mM Imidazole).

Cell lysis by sonication

• Lyse the cell pellet by sonication (Branson) for 10 minutes at 50% amplitude at alternating 2 second intervals, kept on ice.
• Centrifuge the cell lysate at 15000 rpm for 45 minutes.
• Collect the supernatant (soluble fraction).

IMAC

(Immobilised metal ion affinity chromatography (IMAC) was performed to purify the expressed proteins.)

• His-tagged protein is bound to a 1 mL Ni-NTA Superflow Cartridge (Qiagen) by loading the soluble fraction of the cell lysate onto the column.
• Wash with 10 mL of binding buffer (20mm NaH2PO4, 500mM NaCl, 10mM Imidazole).
• Elute with 2 mL of elution buffer (same as binding, but with 500 mM imidazole).
• Collect fractions with SDS-PAGE.

Buffer exchange

Column

• Elutions were analysed with SDS-PAGE and buffer exchanged into PBS pH 8 using Pierce Protein Concentrators PES, 10K MWCO, 2-6 mL (Thermo Scientific).
• Add protein to the column
• Top up column with PBS buffer
• Centrifuge column at 4600 x g for 20 minutes.
• Repeatedly centrifuge, discard flow through, and top up with PBS buffer (pH 8) until dilution factor of 0.1 is reached. That is, there is 1% of the old buffer left in the solution.

Dialysis

• Add 1mL of protein and 1mL of PBS buffer (PH 8) to a 15mL Falcon tube.
• Add 2mL of the solution to a SnakeSkin™ Dialysis Tubing, 10K MWCO, 22 mm.
• Use dialysis tubing clamps (one-piece polypropylene clamp) to further secure the solution inside the snakeskin dialysis tubing.
• Add 500mL of PBS buffer, pH 8, (this is the buffer we want to exchange into) into a 500ml glass beaker.
• Place the dialysis tubing with the solution into the beaker.
• Place the beaker on top of a magnetic stirrer, 75 rpm, and leave overnight.