Team:UNSW Australia/Experiments

Experiments

Cloning

Linearisation pETDuet1 and pRSFDuet1 plasmid backbones using PCR

  • Forward and reverse primers created for the plasmids
  • 2 uL of plasmid + 198 uL of water
Component 50 µl Reaction Final Concentration
Q5 High-Fidelity 2X Master Mix

 25 µl

 1X
10 µM Forward Primer

2.5 µl

0.5 µM

10 µM Reverse Primer

2.5 µl

0.5 µM

Template DNA

2 uL DILUTED

< 1,000 ng

Nuclease-Free Water

18 uL

  
STEP TEMP TIME

Initial Denaturation

98°C

30 seconds

25–35 Cycles

98°C

5–10 seconds

62

10–30 seconds

72°C

2 min

Final Extension

72°C

2 minutes

Hold

4–10°C

  

Plasmid digest (Dpn1 digest)

  • Set-up the reaction mixture: Code:

    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

Agarose gel electrophoresis

  • Combine 100x agarose powder with 1x TAE buffer in a microwavable flask (eg. 1 g of agarose for 100 mL of TAE). The volume of agarose gel will depend on the size of gel you are making.
  • Microwave for 1-2 min until the agarose is completely dissolved (do not overboil the solution). Stop and swirl the flask every 20 seconds and until the solution is as clear as water.
  • Let agarose solution cool down to about 50 C (when you can comfortably hold the flask with your hand), then add 1 uL of RedSafe to the agarose solution.
  • Seal the ends of a gel tray using masking tape. Pour the agarose into the gel tray with a well comb in place. Let the gel sit at room temperature for 20-30 mins until the gel solidifies.
  • Place the gel into the gel box, fill the gel box with 1 x TAE buffer until the gel is covered then remove the well comb.
  • Mix 2 uL of the digest sample with 3 uL of H2O and 1 uL of 6x loading dye.
  • Load 5 uL of 1kb DNA ladder into the first lane of the gel. Load the remaining digest samples into the gel. Remember to include a negative control (non-digested plasmid).
  • Connect the gel box to a power pack and run the gel at 100V for 1 hr. You should be able to see small bubbles rising in the buffer solution immediately after you turn the power pack on.
  • Carefully take the gel tray to the spectrophotometer and analyse the DNA fragments with UV light. We expect to see a single clear band in digested samples, and a smear for the undigested plasmid at a higher position. Smear and a clear band indicates incomplete digestion.

Gibson Assembly

Materials:

  • 5X Isothermal Reaction Mix (6 mL total)
    • 3 mL 1 M Tris-Hcl (pH 7.5)
    • 300 μL 1 M MgCl2
    • 60 μL 100 mM dGTP
    • 60 μL 100 mM dATP
    • 60 μL 100 mM dTTP
    • 60 μL 100 mM dCTP
    • 300 μL 1 M DTT
    • 1.5 g PEG-8000
    • 300 μL 100 mM NAD
    • 360 µL water
    • Store at -20°C.
  • Assembly Master Mix (1.2 mL total)
    • 320 μL 5X Isothermal Master Mix
    • 0.64 μL 10 U/μL T5 exonuclease
    • 20 μL 2 U/μL Phusion DNA Pol
    • 0.16 μL 40 U/μL T4 DNA Ligase
    • 860 μL water
    • Store in 15 µL aliquots at -20°C.
    • PCR or digest your fragment of choice and gel purify
    • If PCR from a methylated DNA template (e.g. propagated plasmid), a DpnI digest can be used to remove the unwanted plasmid. Clean up afterwards.
    • Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.
    • Add 5 μl of DNA to be assembled to the master mixture.
    • The DNA fragments should be in equimolar amounts.
    • Small fragments (<1 kb) must be added in a five times excess
    • You can calculate the quantity of each fragment using their molecular weights.
    • Alternatively, you can use the length of each fragment as a proxy for the molecular weight (assuming similar GC content in all fragments).
    • Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
    • Incubate at 50 °C for 15 to 60 min (60 min is optimal).

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

  • Pick up 5 individual bacterial colonies from each plate that was grown overnight using a pipette tip and dilute each colony into 50 uL of water. Label A, B, C, D, E etc.
  • Create the colony PCR master mix. Determine volumes for the cPCR master mix by multiplying (the number of reactions + 2) by each volume below:
    • 18 uL Nuclease free water
    • 5 uL 5x Taq master mix
    • 0.5 uL 10 uM T7 promoter primer
    • 0.5 uL 10 uM T7 terminator primer
    Example, for 20 colonies, you would add 22 x 18 uL of water, 22 x 5 uL of 5x Taq master mix, 22 x 0.5 uL of forward primer and 22 x 0.5 uL of reverse primer.
  • Add 1 uL of each bacterial dilution and 24 uL of cPCR master mix to a PCR tube.
  • Run PCR with the following instructions (lid at 105 C and volume = 20 uL):
    • 95 C 5:00 mins
    • 95 C 0:30 mins
    • 55 C 0:30 mins
    • 68 C 2:00 mins
    • 68 C 5:00 mins
    • 4 C 0:00 mins (hold)
  • Run PCR products on a 1% agarose gel, TAE running buffer, 100V, 1 hr. 10 uL of PCR product + 2 uL of 6x loading dye, and have 100bp ladder in the first lane.

    • 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.

    Restriction cloning

    • Set-up the reaction

      Restriction Enzyme

      1µl of each enzyme

      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

    Ligation

  • Set up the following reaction in a microcentrifuge tube on ice:

    2 μl

    Component 20 μl Reaction

    T4 DNA Ligase Buffer (10X)*

    Vector DNA

    50 ng

    Insert DNA

    A molar ratio of 1:3 vector to insert should be used

    Nuclease-free water

    to 20 μl

    T4 DNA Ligase

    1 μl
  • Gently mix the reaction by pipetting up and down and microfuge briefly.
  • For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
  • Heat inactivate at 65°C for 10 minutes.
  • Chill on ice and transform 1-5 μl of the reaction into 25 μl competent cells.

    • Miniprep

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

    Protein Expression and Purification

    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.