Team:Sydney Australia/Protocols

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Making Agar Plates

  1. Prepare LB-Agar for autoclave: 10 g tryptone, 5 g yeast extract and 5 g of NaCl per litre.
  2. Autoclave.
    If it is still molten when collected, it can be left in a 60°C water bath to cool down for poouring. Otherwise, if it has solidified, it needs to be microwaved until molten, then left in a 60°C water bath to cool to the correct temperature.
  3. Once agar is molten, mix carefully. Add antibiotics and any other desired additives.
  4. Pour plates carefully.
  5. Allow to set for approximately 10 min.
  6. Invert plates and allow to dry for 20-30 min.
  7. Store plates at 4°C

Inoculating Liquid Cultures

It is important to practise sterile technique when growing bacterial cultures. In our project, we grew E. coli. cells.

  1. Prepare LB broth (10 g tryptone, 5 g yeast extract, 10 g NaCl per litre, pH 7.5). Autoclave.
  2. Prepare an aliquot of appropriate size including antibiotics and any other desired additives.
  3. Use a sterilised loop or pipette tip to pick up some of the culture that you wish to inoculate - this can be from an agar plate, a liquid broth or glycerol stock
  4. Add to the prepared aliquot of LB
  5. Grow at 37°C with shaking (200 rpm)

Plasmid Miniprep Using Bioline ISOLATE II Plasmid Mini Kit

All plasmids were miniprepped using the manufacturer’s protocol (the following protocol contains lab-specific details and notes):

  1. Grow bacterial cells.
    Inoculate cells containing the desired plasmid into 5 mL LB culture containing the appropriate antibiotic, and grow at 37°C overnight, with shaking (200 rpm).
  2. Harvest bacterial cells.
    In the morning, transfer cultures to 15 mL Falcon tubes and centrifuge for 15 minutes at 35 000 x g.
    Discard supernatant and remove as much liquid as possible.
  3. Lyse cells
    • Add 250 μL Resuspension Buffer P1 and resuspend cell pellet by vortexing or pipetting up and down.
    • Add 250 μL Lysis Buffer P2.
      Mix gently by inverting tube 6-8 times.
    • Incubate at room temperature for up to 5 min or until lysate appears clear.
      We found that incubating for 5 min at room temperature at this step helps to increase yield.
    • Add 300 uL Neutralization Buffer P3.
      Mix thoroughly by inverting tube 6-8 times.
  4. Clarification of lysate.
    Centrifuge 5 min at 11,000 x g at room temperature.
  5. Bind DNA.
    Place ISOLATE II Plasmid Mini Spin Column in a 2 mL Collection Tube (supplied).
    Decant or pipette a maximum of 750 μL of clarified sample supernatant onto column.
    Centrifuge 1 min at 11,000 x g and discard flow-through.
  6. Wash silica membrane.
    If plasmid DNA is prepared from host strains containing high levels of nucleases, and extra wash with Wash Buffer PW1 is strongly recommended.
    (Optional) Add 500 μL Wash Buffer PW1 preheated to 50°C. We always carried out this step.
    Centrifuge 1 min at 11,000 x g.
    Discard flow-through and reuse Collection Tube.
  7. Dry silica membrane.
    Centrifuge 2 min at 11,000 x g, to remove residual ethanol.
    Place ISOLATE II Plasmid Mini Spin Column in a 1.5 mL microcentrifuge tube (not supplied).
  8. Elute DNA.
    Add 50 μL Elution Buffer P (we used homemade elution buffer) directly onto the centre of the silica membrane.
    Incubate at room temperature for 1 min.
    Centrifuge 1 min at 11,000 x g.

Making and Running Agarose Gels

  1. Set up gel tank as appropriate
  2. Weigh out an appropriate quantity of agarose for the volume of gel required. For DNA <1 kb, we generally used 1.5% agarose. For DNA >1kb, we generally used 0.8% agarose.
  3. Add an appropriate volume of buffer. For DNA <1kb, we generally used 0.5x Tris-borate EDTA buffer (TBE). For DNA >1kb, we generally used 1x Tris-acetate EDTA buffer (TAE).
  4. Microwave until molten. The solution should be clear.
  5. Allow to sit on bench until it is cool enough to touch. This can be sped up by running the container under cold water.
  6. Seal the gel apparatus by adding a little molten agarose to the edges where leakage may occur, and allowing this small amount of agarose to set.
  7. Pour gel and add well combs.
  8. Allow to set.
  9. Fill the tank with the same running buffer used to cast the gel so that the buffer level just covers the gel.
  10. Load samples with an appropriate loading dye. Samples can also be pre-stained with GelGreen or similar by adding the stain into the sample being loaded.
  11. Run at an appropriate voltage, generally 100-200 V depending on the agarose concentration, size of DNA being resolved and the gel apparatus.
  12. Post-stain in GelGreen or similar, if the samples were not pre-stained. This stain generally takes about one hour at room temperature or 40 min at 50°C.
  13. Image under UV light.

PCR Amplification

This is a generic protocol for PCR reactions. It has been adapted in some of our other protocols for specific purposes, such as colony PCR and error-prone PCR.

  1. Prepare master mix.
    For one 25 μL reaction:
    2. Component Volume (μL)
    Thermopol 10x buffer 2.5
    Sterile milliQ water 20
    dNTPs (10 mM) 0.5
    FWD primer (50 μM) 0.25
    REV primer (50 μM) 0.25
    Polymerase 0.25
  2. Aliquot master mix into PCR tubes.
  3. Add 1 μL of DNA template to each tube. For concentrated plasmid stocks, this may need to be diluted first as too much template can carry over into the product.
  4. Carry out thermocycling with appropriate parameters. For example, a typical cycle for Taq polymerase might involve:
    • Initial denaturation at 95°C
    • 30 cycles of:
      • Denaturation at 95°C for 30 sec
      • Annealing at 60°C for 30 sec
      • Extension at 72°C for 1 min
    • Final extension at 72°C for 10 min
    • Hold at 15°C

    Note that annealing temperature should be optimised for the primers. Extension time should also be adjusted depending on the size of the PCR product and the polymerase. For Taq polymerase, 1 min per kb is generally appropriate.
  5. Check PCR product on agarose gel.

Cloning via Restriction Digestion and Ligation

All cloning work was carried out via restriction digestion and ligation. Typically, 250 ng – 1 µg of plasmid DNA and 250 – 500 ng of insert DNA was digested.

Restriction Digestion

  1. Set up digestion mix with the following components:
    • 10x CutSmart Buffer (NEB)
    • Nuclease-free water to a final volume of 20 to 30 uL, depending on the amount of DNA added.
    • DNA
    • Restriction enzyme (typically 2 uL for cloning purposes)
  2. Digest for ~2 hours at optimal temperature for enzyme function (typically 37°C).
  3. Gel purify plasmid backbone and column purify inserts using the DNA Purification Using Bioline ISOLATE II PCR and Gel Kit. To prepare plasmid sample for gel purification, run entire digest on a 1% agarose gel in TAE buffer, pre-staining with GelGreen Nucleic Acid Gel Stain.
  4. Quantitate yields of purified plasmid and insert using a NanoDrop.

Ligation

  1. Calculate amount of plasmid and insert required to obtain a 1 to 3 molar ratio of plasmid to insert for ligation.
  2. Set up ligation mixes with the following components:
    • 10x T4 DNA ligase reaction buffer
    • Nuclease-free water to a final volume of 10 uL
    • Plasmid DNA
    • Insert DNA
    • 1 µL T4 DNA ligase (concentrated stock from NEB)
  3. Set up a ligation control containing the same amount of the above components, except without insert DNA.
  4. Retain some of the purified digested plasmid to be used as a digestion control.
  5. Incubate ligations at 4°C overnight or room temperature for 1 hour.
  6. Transform into chemically competent E. coli by heat shock.

Preparation of Chemically Competent Cells

We produced chemically competent cells using rubidium chloride and calcium chloride.

  1. Prepare RF1 solution:
    2. Component Amount Final conc.
    Reverse Osmosis Water 500 mL NA
    RbCl 6.06 g 100mM
    MnCl2 4.95 g 50 mM
    K Acetate 1.47 g 30 mM
    CaCl2 0.74 g 10 mM
    Glycerol 75 g 15%

    Adjust pH to 5.8 with conc. acetic acid. pH will change very quickly and only requires ~ 10µL of acetic acid. Sterilize by filtration into an autoclaved media bottle.
  2. Prepare RF2 solution:
    3. Component Amount Final conc.
    Reverse Osmosis Water 500 mL NA
    MOPS 1.06 g 10mM
    RbCl 0.6 g 10 mM
    CaCl2 5.52 g 75 mM
    Glycerol 75 g 15%

    Adjust pH to 6.8 with NaOH or HCl, as appropriate. pH will change very quickly and only requires ~ 10µL of acetic acid. Sterilize by filtration into an autoclaved media bottle.
  3. Streak out the desired strain of E. coli. onto plain LB agar and incubate at 37°C overnight.
  4. Inoculate 5 mL of plain LB and incubate overnight at 37°C with shaking (200 rpm).
  5. Inoculate 100 mL plain LB with 3 mL of the overnight culture to give an OD600 of approximately 0.05.
  6. Grow cells at 37°C with shaking (200 rpm) until the OD600 of approximately 0.5.
  7. Pellet cells at 4°C and 3000xg for 10 min and pour off the supernatant.
  8. Resuspend the pellet gently in RF1 solution. Use 33 mL RF1 solution for each 100 mL of culture used.
  9. Incubate on ice for 1 h.
  10. Pellet cells at 4°C and 3000xg for 10 min and pour off the supernatant.
  11. Resuspend the pellet gently in RF2 solution. Use 8 mL RF2 solution for each 100 mL of culture used.
  12. Incubate on ice for 15 min.
  13. Aliquot and freeze at 80°C. Test the transformation efficiency using a known amount of plasmid standard.

Heat Shock Transformation of Chemically Component E. coli.

E. coli. rendered chemically competent through treatment with calcium or rubidium chloride can be transformed with circularised DNA from isolated plasmids or from ligation products.

  1. Thaw competent cells (prepared as such ) on ice.
  2. Add 50 uL of competent cells to a pre-chilled 1.5 mL Eppendorf tube.
  3. Add transformant DNA: 1 uL of isolated plasmid, or 3 uL of ligation/digestion products.
    • We recommend doing a negative control (no DNA), a positive control (plasmid with correct resistance), a digestion control (no insert) and a ligation control (no insert, ligation performed)
    • Note: do not vortex competent cells – to mix, flick the tube gently
  4. Incubate on ice for 20 min.
  5. Heat-shock at 42°C for 45 seconds and rest on ice for 2 minutes.
  6. Add 1000 uL of LB- and shake at 37°C for 1 hour.
  7. Plate out 100 uL of the cells on selective LB and then centrifuge at 11,000 g for 1 minute to pellet the remaining cells. Pour off the supernatant, resuspend the cells in the remaining liquid and plate out on selective LB.
  8. Incubate plates at 37°C for a minimum of 12 hours for transformant colonies to appear.

DNA Purification Using Bioline ISOLATE II PCR and Gel Kit

Purification of PCR products, restriction endonuclease digests etc. were carried out using the “PCR Clean-Up” protocol, and purification of DNA from agarose gels were carried out using the “DNA Extraction From Agarose Gels” protocol, both provided with the kit.
The protocols below have been adapted from the manufacturer’s protocol, with lab-specific details and notes.

PCR Clean-up

  1. Sample preparation
    For volumes <30 μL, adjust volume to 50-100 μL with water.
    Mix 1 volume of sample with 2 volumes of Binding Buffer CB.
  2. Bind DNA
    Place an ISOLATE II PCR and Gel Column in a 2mL Collection Tube and load sample.
    Centrifuge 30s at 11,000 x g and discard flow-through.
    Reuse collection tube for step 3.
  3. Wash silica membrane.
    Add 700 μL Wash Buffer CW to ISOLATE II PCR and Gel Column.
    Centrifuge 30s at 11,000 x g.
    Discard flow-through and place column back into Collection Tube.
    Recommended: Repeat washing step to minimize chaotropic salt carry-over. This step was always carried out.
  4. Dry silica membrane.
    Centrifuge 1 min at 11,000 x g, to remove residual ethanol.
    Place ISOLATE II PCR and Gel Column in a 1.5 mL microcentrifuge tube (not supplied).
  5. Elute DNA
    Add 15-30 μL Elution Buffer C directly onto silica membrane.
    Incubate at room temperature for 1 min.
    Centrifuge 1 min at 11,000 x g.

DNA Extraction From Agarose Gels

  1. Excise and dissolve gel slice
    Using a clean scalpel excise DNA fragment from gel.
    Remove excess agarose, determine weight of gel slice and transfer into a clean tube.
    Our DNA samples were always prestained with GelGreenTM Nucleic Acid Gel Stain, before running samples on an agarose gel made in TAE buffer. We found that using a hand-held UV light in a dark room and wearing orange-filtered glasses was effective in visualising the DNA bands on the gel.
    Add 200 μL Binding Buffer CB per 100 mg of 2% agarose gel*.
    *For gels containing >2% agarose, double the volume of Binding Buffer CB.
    Incubate sample at 50°C for 5-10 min, vortexing sample briefly every 2-3 min until gel slice is completely dissolved.
  2. Bind DNA
    Place ISOLATE II PCR and Gel Column in a 2 mL Collection Tube and load sample.
    Centrifuge 30s at 11,000 x g and discard flow-through.
    Reuse collection tube for step 3.
  3. Wash silica membrane.
    Add 700 μL Wash Buffer CW to ISOLATE II PCR and Gel Column.
    Centrifuge 30s at 11,000 x g.
    Discard flow-through and place column back into Collection Tube.
    Recommended: Repeat washing step to minimize chaotropic salt carry-over. This step was always carried out.
  4. Dry silica membrane.
    Centrifuge 1 min at 11,000 x g, to remove residual ethanol.
    Place ISOLATE II PCR and Gel Column in a 1.5 mL microcentrifuge tube (not supplied).
  5. Elute DNA
    Add 15-30 μL Elution Buffer C directly onto silica membrane.
    Incubate at room temperature for 1 min.
    Centrifuge 1 min at 11,000 x g.

Screening Recombinant Clones Via Colony PCR and Diagnostic Digest

Recombinant clones were screened using colony PCR, before positive colonies were picked for diagnostic digest.

Colony PCR

  1. Make a PCR master mix with the following components:
    For 1 reaction:
    Component Volume (μL)
    Thermopol 10x buffer 2.5
    Sterile milliQ water 20.875
    dNTPs (10 mM) 0.5
    FWD primer* (50 μM) 0.5
    REV primer* (50 μM) 0.5
    Taq polymerase 0.125

    *Primers can be designed in any way that will enable the detection of the insert in the plasmid. Typically, we used a reverse primer in the plasmid backbone and a forward primer in the insert.
  2. Using a P10 tip or equivalent, pick a single colony and put a few cells into a PCR tube by touching the tip onto the bottom of the tube.
  3. Streak the remaining cells onto a patch plate (LB agar plate with the appropriate antibiotic, divided into a grid).
  4. Repeat steps 2 and 3, picking a mix of large and small colonies (as uptake of the recombinant plasmid could slow cell growth, resulting in smaller colonies). We typically pick 20 colonies for colony PCR screening.
  5. Add 25 µL of master mix into each tube.
  6. Flick and spin down the PCR tubes, and run PCR with the following thermocycler conditions:
    • 95°C for 30 seconds
    • 95°C for 15-30 seconds, 45-68°C for 15-60 seconds, 68°C for 1 minute/kb (30 cycles)
    • 68°C for 5 minutes
  7. Run PCR products on an agarose gel to check whether the expected band has been amplified

Error-prone PCR

Error-prone PCR generates random mutations in the amplified product of a target sequence. We did this by addition of manganese chloride to the reaction mix to cause Taq polymerase to lose its fidelity.

  1. Thaw all reagents
  2. Prepare master mix containing 10x Thermopol® buffer (NEB), sterile milliQ water, dNTPs, forward primer, reverse primer and manganese chloride*.
    3. Component Amount (μL per 50 μL reaction)
    10x buffer 5
    Sterile milliQ water 40.5
    dNTPs (10 mM) 1
    FWD primer (50 μM) 0.5
    REV primer (50 μM) 0.5
    Manganese chloride* (5-50 mM. whatever is 50x desired final concentration) 1
  3. Aliquot into reaction tubes.
  4. Add 1 μL DNA template to each tube.
  5. Thermocycle: 95°C initial denaturation for 5 min,15 cycles** of: 95°C denaturation for 1 min, 60°C annealing for 1 min, 72°C extension for 3 min***, 72°C final extension for 10 min, 15°C hold.
*Final concentrations of MnCl2 at 0.5 mM significantly reduce PCR efficiency. Concentrations 0.1 – 0.5 mM give reasonable mutation frequencies.
**Fewer cycles than a regular PCR, to prevent overamplification of short sequences produced by mispriming [1]
***Long extension time is important to reduce selective amplification of shorter, undesirable sequences produced by mispriming [1]

1. Wilson, D.S. and A.D. Keefe, Random Mutagenesis by PCR, in Current Protocols in Molecular Biology. 2001, John Wiley & Sons, Inc.

Spectrophotometric Analysis of Chromoproteins

This method was used to analyse the different mutants of amilCP that were developed during this project.

  1. Inoculate freshly transformed cells into 100mL LB with appropriate antibiotics, and incubate at 37˚C with shaking for 1-3 days until colour is visible.
  2. Centrifuge 50mL for 20 minutes at 35000xg.
  3. Resuspend the pellet in 0.5mL 1X TE buffer with 0.5% Triton X-100, and transfer to a 2mL bead beating tube prepared earlier with glass beads*.
  4. Freeze at -20˚C until frozen.
  5. Thaw in a warm water bath.
  6. Repeat steps 4 and 5**.
  7. Place on ice for 5 minutes.
  8. Bead beat for 30 seconds.
  9. Incubate on ice for 1 minute.Repeat steps 9 and 10 twice more (bead beat for 1.5 minutes in total).
  10. Centrifuge at 4˚C for 5 minutes at 15000xg.
  11. Transfer the supernatant to a new tube.
  12. Measure the protein concentration of each sample using a NanoDrop or other spectrophotometer at 280nm, and dilute each sample to match the sample with the lowest protein concentration.
  13. Perform a spectrum scan from 300 to 750nm using a spectrophotometer.
* We used one large glass bead, 3 scoops of small beads and 3 scoops of very small beads. More beads than usual are necessary for a higher chromoprotein yield.
** Repeated slow freezing and thawing helps to break open the cells.

IPTG Induction of Protein Expression

Genes placed under the control of the T7 promoter can be inducibly expressed through the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG). We used this for expression of genes in recombinant E. coli. of the strain BL21 (DE3) with helper plasmid pLysS or pGro7.

  1. Inoculate E. coli. into 5 mL of LB with appropriate antibiotics and shake at 37°C for 16 hours (overnight).
  2. Add the 5 mL culture to 50 mL of LB with appropriate antibiotics. For the helper plasmid pGro7, supplement the LB with arabinose to a final concentration of 0.5 mg/mL to induce expression.
  3. Shake at 37°C until the OD600 reaches a value of 0.5. Add IPTG to a final concentration of 1 mM and leave at 25°C for 6 hours with shaking.
  4. Centrifuge the cells at 3,500 g for 15 minutes to pellet the cells. Pour off the supernatant and wash thrice with 5 mL of PBS.

Purification of His6-Tagged Proteins

Proteins expressed with a consecutive series of 6 histidines, or His6-Tagged, at the N or C terminus can be purified based on the affinity of histidine residues for divalent metal cations such as Ni2+ and Co2+. By passing cellular lysates through beads coated with these ions, the proteins of interest can be captured whilst the remaining cellular proteins are washed off. The tagged proteins can then eluted by the addition of a solution with a high concentration of imidazole (~300 mM) which competitively binds the nickel or cobalt, forcing the protein off.

GE Healthcare HisTrap HP 1 mL Column Protocol: Ni2+ beads

  1. Resuspend a cell pellet with the His6-Tagged protein of interest using 1 mL of binding buffer (20 mM sodium phosphate, 0.5 M sodium chloride, 5 mM imidazole, pH 7.4).
    • Note: a higher concentration of imidazole (between 20 mM and 40 mM) can be used to reduce non-specific binding of cellular proteins to the Ni2+ ions.
    • Note: membrane proteins and protein expressed in inclusion bodies can be purified by adding 8 M urea to all buffers as a denaturant. Refolding of the protein can be performed on or off the column by gradually reducing the amount of denaturant present either using a urea wash gradient for on-column refolding, or using dialysis for off-column refolding [not verified experimentally in this project].
  2. Add the cell suspension to bead beater tubes containing a range of glass beads of various sizes. Bead-beat the cells for 30 seconds. Transfer the mixture immediately to ice.
  3. Spin for 20 minutes at 15,000 g at 4°C. Take the supernatant and aliquot out 20 uL as an unpurified control.
  4. Wash a HisTrap HP column with 5 column volumes (1 column volume = 1 mL) distilled water and equilibrate with 5 column volumes of binding buffer.
    • Note: a flow rate of 1 mL/min is recommended. This can be achieved using an automated syringe pusher.
  5. Perform a blank run by washing the column with 5 column volumes of distilled water, followed by 5 column volumes of elution buffer (20 mM sodium phosphate, 0.5 M sodium chloride, 500 mM imidazole, pH 7.4) and 10 column volumes of binding buffer.
  6. Apply the cell lysate and wash with 15 column volumes of binding buffer. Collect the flowthrough in 3 x 5 mL fractions.
  7. Elute the protein using elution buffer.
    • For a one-step elution, elute with 5 mL elution buffer and collect 0.5 mL fractions.
    • For a gradient elution, elute with 2 mL elution buffers with varying imidazole concentrations (10, 20, 50, 100, 200, 300 and 500 mM) and collect 1 mL fractions.
  8. Quantitate the protein fractions obtained and run on SDS-PAGE to verify purity (protocol here)

Dynabeads His-Tag Isolation

  1. Resuspend a cell pellet with the His6-Tagged protein of interest using 600 μL of binding buffer (50 mM sodium phosphate, 300 mM NaCl, 0.01% Tween-20, pH 8.0).
  2. Add the cell suspension to bead beater tubes containing a range of glass beads of various sizes. Bead-beat the cells for 30 seconds. Transfer the mixture immediately to ice.
  3. Spin for 20 minutes at 15,000 g at 4°C. Take the supernatant and aliquot out 20 uL as an unpurified control.
  4. Resuspend Dynabeads by vortexing for 45seconds and transfer 50 uL to a 1.5 mL Eppendorf tube. Place the tube on the Dynamax magnet for 2 minutes.
    • Note: any other magnet will serve the same purpose. If no magnet is available, then spin down using a microcentrifuge.
    • Note: all procedures from here onwards were performed at 4°C.
    • Note: it’s recommended that all supernatants are kept for analysis.
  5. Remove the supernatant and add the cell lysate to the beads. Mix well and incubate on a shaker for 10 minutes.
    • Note: avoid mixing the beads and sample with a pipette – flick or drag against racks to avoid loss of beads and protein.
  6. Place the tube on the magnet for 2 minutes and take off the supernatant.
  7. Wash beads 4 times with 300 uL binding buffer by mixing the beads well, placing tube on magnet for 2 minutes and taking off the supernatant.
  8. Add 100 uL elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 300 mM imidazole, 0.01% Tween-20, pH 8.0) to the beads.
  9. Mix well and incubate on a shaker for 5 minutes.
  10. Place the tube on the magnet for 2 minutes and take off supernatant as eluted protein.
  11. Quantitate the protein fractions obtained and run on SDS-PAGE to verify purity

Dialysis

Dialysis can be used to remove excess salts, such as imidazole, and also help to concentrate the protein sample.

  1. Prepare dialysis buffer - 50 mM sodium phosphate, 5% glycerol and 0.01% Tween-20, pH 8.0.
    Note: glycerol helps to absorb water to concentrate the protein. It can be left out of the buffer if the protein is already sufficiently concentrated.
  2. Load sample into Slide-A-Lyser Dialysis Casette (Thermo Fisher).
    Note: We learnt from experience that it is very difficult to load and retrieve samples where the volume is <0.5 mL. Ideally a larger volume should be used.
  3. Dialyse overnight.
  4. Retrieve sample from dialysis casette.

Making and Running SDS-PAGE Gels and Preparation of Cell Lysates

Cellular proteins can be isolated through physical and chemical lysis or by other means of disrupting cell membranes. These proteins can then be separated electrophoretically through denaturing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) as proteins with different mobility, dependent on length and charge, will separate in size-dependent manner.

Cellular Lysis

  1. Resuspend a cell pellet in Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0)
  2. Add the cell suspension to bead beater tubes containing a range of glass beads of various sizes. Bead-beat the cells for 30 seconds. Transfer the mixture immediately to ice.
    • Note: if the protein is not found in the soluble fraction, add SDS to a final concentration of 2% and heat at 95°C.
  3. Quantitate the concentration of protein samples using NanoDrop2000.

SDS-PAGE

  1. Prepare resolving gels (0.375 M Tris-HCl, 12% acrylamide, 0.1% SDS, 0.05% ammonium persulfate and 0.05% tetramethylethylenediamine (TMED)) and pour into gel formers.
  2. Once the resolving gel is set, prepare stacking gels (0.125 M Tris-HCl, 4% acrylamide, 0.1% SDS, 0.05% ammonium persulfate and 0.1% TMED) and pour on top of the resolving gel. Add well combs on top of each gel.
  3. Once the stacking gel is set, place the gels in a running tank, and add running buffer (25 mM Tris, 0.20 M glycine, 0.1% SDS).
  4. Remove well combs and load samples into wells.
  5. Run at 200 V for 1 hour, or until the dye front reaches the end of the gel.
  6. Stain in Commassie Brilliant Blue dye (0.1% Commassie Brilliant Blue R-250, 50% methanol and 10% acetic acid) for 30 minutes with shaking at room temperature.
  7. Destain using high destain (40% methanol and 10% acetic acid) for 30 minutes with shaking at room temperature, followed by low destain (10% methanol and 10% acetic acid) overnight.

Electrophoretic Mobility Shift Assay (EMSA)

This assay tests for the binding of a certain protein to a certain segment of DNA through assessing the migration of the DNA through an electrophoresis gel. Protein-bound DNA should demonstrate band retardation. For our experiments, we used EtnR1 protein purified by his-tag using Dynabeads® followed by overnight dialysis.

  1. Prepare 5x binding buffer (100 mM KCl, 0.5 mM dithiothreitol, 50 mM Tris, pH 7.4). This buffer was the one that we used, however, it should be further optimised for each individual protein-DNA combination.
  2. Mix 100 ng of target DNA with 5x binding buffer (to 1x concentration) and protein (5-10 μg).
  3. Incubate at room temperature for 30 min – 1 h.
  4. Prepare 3% agarose gel with wells cast in 1% agarose in 0.5x TBE buffer (Tris borate EDTA; 45 mM Tris-borate, 1 mM EDTA, pH 8.0).
    This is because high percentage agarose gels are very brittle. Thus it is necessary to first cast the gel without wells in 3% agarose and allow to set. Then, cut away 2 cm from one end and add 1% agarose and well combs. Allow to set.
  5. Load samples using loading solution (no SDS) containing 4% Ficoll, 40% glycerol. Add regular loading dye to an appropriate DNA ladder to track the progress of the gel.
  6. Run gel at 150-200 V.
  7. Post stain in GelGreenTM (Biotium) at 50˚C for 1 h or until bands are visible. This can be extended to an overnight stain, but the DNA bands tend to diffuse and become less sharp.
  8. Image under UV light.

School of Life and Environmental Sciences
The University of Sydney
City Road, Darlington
2006, New South Wales, Sydney, Australia