Difference between revisions of "Team:UNSW Australia/Experiments"

Latest revision as of 07:55, 23 November 2018

This page includes all the experimental protocols used by the UNSW iGEM team. Please click on each heading to view the protocols or view our weekly progress on the notebook page. Alternatively, to see the detailed experimental introduction, results and discussion, please visit the lab overview page.

DNA Cloning

Linearisation pETDuet1 and pRSFDuet1 Plasmid Backbones Using PCR

Forward and reverse primers created for the plasmids.
2 µL of plasmid + 198 µL of water.
PCR amplification, according to the table below:

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 µL diluted	< 1,000 ng
Nuclease-Free Water	18 µL
Step	Temperature	Time
Initial Denaturation	98°C	30 seconds
25–35 Cycles	98°C	5–10 seconds
	62°C	10–30 seconds
	72°C	2 minutes
Final Extension	72°C	2 minutes
Hold	4–10°C

Plasmid Digest (Dpn1 Digest)

Set-up the reaction mixture:

Restriction Enzyme

1 µL

DNA

1 µg

10X Cutsmart

5 µL (1X)

Total Reaction Volume

50 µL
Incubate for 1 hr at 37°C.
Heat inactivate at 80°C for 20 minutes.

Agarose Gel Electrophoresis

Combine 1 g of agarose powder with 100 mL of 1x TAE buffer in a microwavable flask.
Microwave for 1-2 minutes until the agarose is completely dissolved. Avoid over-boiling, and stop to swirl the flask every 20-30 seconds until the solution is clear.
Allow the solution to cool down until you can comfortably hold the flask with your hand, then add 1 µL of RedSafe.
Seal the ends of a gel tray and pour the solution into the tray with a well comb in place. Let the solution sit at room temperature for 20-30 minutes until it solidifies into a gel.
Place the gel into the gel tank, and fill the tank with 1X TAE buffer until the gel is covered. Remove the well comb.
Mix 2 µL of the DNA sample with 3 µL of H₂O and 1 µL of 6X loading dye.
Load DNA ladder into the first well of the gel, followed by your DNA samples.
Connect the gel tank to a power pack and run the gel at 100 V for 1 hr.
Carefully take the gel tray to a Gel Doc for imaging.

Gibson Assembly

Materials:

5X Isothermal Reaction Mix (6 mL total, Store at -20°C):

1 M Tris-HCl (pH 7.5)	3 mL
1 M MgCl₂	300 µL
100 mM dGTP	60 µL
100 mM dATP	60 µL
100 mM dTTP	60 µL
100 mM dCTP	60 µL
1 M DTT	300 µL
PEG-8000	1.5 g
100 mM NAD	300 µL
H₂O	360 µL

Assembly Master Mix (1.2 mL total, store in 15 µL aliquots at -20°C.):

5X Isothermal Master Mix	320 µL
510 U/µL T5 exonuclease	0.64 µL
2 U/µL Phusion DNA Polymerase	20 µL
40 U/µL T4 DNA Ligase	0.16 µL
H₂O	860 µL

Method:

PCR amplify or digest your fragments of choice and gel purify.
If PCR from a methylated DNA template (e.g. linearised plasmid), a DpnI digest can be used to remove the unwanted template plasmid. Clean up afterwards.
Thaw a 15 µL assembly mixture aliquot and keep on ice until ready to be used.
Nanodrop your DNA fragments. Prepare 50-100 ng of vector with 2-3 fold excess of insert in nuclease-free water to a total volume of 5 µL.
Add 15 µL assembly mixture to the DNA mixture, for a total reaction volume of 20 µL.
Incubate at 50°C for 15 to 60 minutes (60 minutes is optimal).

Heat Shock Transformation

Incubate 50 ng of plasmid construct with 25 µL of chemically competent E. coli T7 express or DH5α on ice for 30 minutes.
Heat shock the cells for 45 seconds at 42°C and place 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 appropriate antibiotics at working concentration and grow at 37°C overnight.

Colony PCR

Pick up individual bacterial colonies from an agar plate that was grown overnight using a pipette tip, and dilute each colony into 50 µL of water.
Prepare the PCR master mix by multiplying the volume for each component shown below (for 1 reaction) by the number of colonies to sample +1 (i.e. number of reactions plus 1 excess).
- 18 µL nuclease free water.
- 5 µL 5X Taq master mix.
- 0.5 µL 10 µM T7 promotor primer
- 0.5 µL 10 µM T7 terminator primer
Add 1 µL of each diluted colony and 24 µL of PCR master mix to a PCR tube.
Run PCR with the following steps (lid at 105°C and volume = 20 µL):
- Step 1: 95°C for 5 minutes
- Step 2: 95°C for 30 seconds
- Step 3: 55°C for 30 seconds
- Step 4: 68°C for 2 minutess
- Repeat 25-35 cycles of steps 2-4
- Step 5: 68°C 5 minutes
- Step 6: Hold at 4°C
Run PCR products on a 1 % agarose gel and image for analysis.

Sequencing

Transfer 10 µL of purified plasmid sample (50-100 ng/µL) to a PCR tube. Add 5 µL of one primer (forward or reverse).
Request sequencing at the UNSW Centre for Genomics via the on-line portal.
Label PCR tubes carefully with numbers as per the on-line order.
Take PCR tubes to the Ramaciotti Centre for Genomics Lvl 2 (at UNSW, Sydney, Australia), and store the samples in the fridge provided.
Sanger sequencing is carried out by the Centre staff following the provided protocol.

Restriction Cloning

Set-up the reaction mixture:

Restriction Enzyme

1 µL of each enzyme

DNA

1 µg

10X Cutsmart

5 µL (1X)

Total Reaction Volume

50 µL
Incubate for 1 hr at 37°C.
Heat inactivate at 80°C for 20 minutes.

Ligation

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

Component	20 µL Reaction
T4 DNA Ligase Buffer (10X)*	2 µL
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 Cat No./ID: 27104. No changes were made.

Protein Production

Starter Culture

One colony was selected from the plate grown overnight and grown in 2 mL of LB containing appropriate antibiotic at 37°C and 200 rpm 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 an absorbance reading of 0.6, add 1mM of IPTG to induce expression of the protein.
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 (20 mM NaH₂PO₄, 500 mM NaCl, 10 mM Imidazole).

Cell Lysis by Sonication

Lyse the cell pellet by sonication (Branson) for 10 minutes at 50% amplitude, 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 (20 mM NaH₂PO₄, 500 mM NaCl, 10 mM Imidazole).
Elute with 2 mL of elution buffer (20 mM NaH₂PO₄, 500 mM NaCl, 500 mM Imidazole).
Analyse 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 to 6 mL 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 0.01 is reached. That is, until there is 1% of the old buffer left in the solution.

Dialysis

Add 1 mL of protein and 1 mL of PBS buffer (pH 8) to a 15 mL Falcon tube.
Add 2 mL 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 500 mL of PBS buffer, pH 8, (this is the buffer we want to exchange into) into a 500 mL 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.

Western Blot

Materials

NuPAGE Bis-Tris gel
NuPAGE MES running buffer
Mini iBlotTM stack
TBS-T:
1X TBS with 0.1 % Tween20
Blocking Solution:
5 % skim milk in TBS-T
Antibody Solution:
1:2000 dilution of HRP conjugated anti-His-tag antibody in TBS-T + 1 % BSA
Chemiluminescent HRP substrate

Sample Preparation

Add reducing buffer to the bacterial lysates.
Heat at 95°C for 5 minutes.

SDS-Page Gel

Remove the NuPAGE gel from its packaging and peel off the plastic strip from its base.
Place the gel inside the tank, and fill with NuPAGE MES running buffer.
Load 5 µL of the protein standards ladder into the first well.
Load up to 20 µL of each lysate sample into the wells.
Connect the gel tank to a power pack, and run at 160 V for 40 minutes.

Protein Transfer

Remove and rinse the gel in water.
Inside an iBlot Transfer Device, assemble the mini stack with the gel inside.
Run at 20 V for 7 minutes.

Blocking

Incubate the membrane for 1-2 hours in blocking solution at room temperature, shaking.

Antibody Staining

Incubate the membrane in antibody solution either at 4°C overnight, or at room temperature for 2 hours.
Wash the membrane in TBS-T three times for 10 minutes per wash at room temperature, shaking.

Detection

Remove membrane from the last wash and place in chemiluminescent image analyser.
Prepare HRP substrate according to manufacturer's instructions and add to the membrane.
Image.

Assembly

Catcher and Tag Assembly

Purified IaaH fused to SpyTag (IaaH-SpyTag) and prefoldin proteins fused to SpyCatcher (aPFD-SpyCatcher, gPFD-SpyCatcher and SpyCatcher-gPFD-SpyCatcher) were obtained.
IaaH-SpyTag was mixed with either aPFD-SpyCatcher, gPFD-SpyCatcher and SpyCatcher-gPFD-SpyCatcher at a concentration of 3 µM and 15 µM respectively in a total volume of 250 µL in PBS pH 8, and incubated at room temperature.
After 0, 10, 20 and 30 minutes of incubation, a 10 µL sample was obtained.
5 µL of 4X Bolt LDS sample buffer was added to each sample, and then boiled for 10 minutes at 95°C to cease SpyCatcher reactivity whilst preserving any covalent interactions.
The samples were then examined via SDS-PAGE.

Size Exclusion Chromotography (SEC)

We would like to thank Hélène Lebhar of the UNSW Recombinant Proteins facility for conducting SEC experiments for us.

TEM

Materials

Protein sample (ideally in water or low salt buffer)
Milli-Q water
2 % uranyl acetate
Parafilm
Self-closing forceps

Staining

Glow discharge the carbon-coated grids (carbon-coated side up) to render them hydrophilic.
Place the grids (carbon-coated side up) on parafilm.
Place 7 µL of sample on a grid.
Let the grid stain for 10 minutes.
Using a pasteur pipette, transfer 5 droplets of Milli-Q water on a piece parafilm (for each grid).
After the 10 minutes of staining, pick up grid with the forceps and wash residue salts by passing the grid through the five droplets of water.
In a fume hood, place one droplet of 2 % uranyl acetate on a piece of parafilm (for each grid).
Wear appropriate PPE to handle uranium; do not work with uranium outside the fume hood.
Place the grid on the uranyl acetate drop (carbon-coated side down) and stain for 5-7 minutes.
Pick the grid up with the forceps.
Remove excess uranyl acetate by touching one side of the grid with filter paper for a few seconds, then briefly touch the other side of the grid with the filter paper.
Let the grid dry for a few minutes before transferring back it to its box.
The grid can be fully dried in a oven if imaging the sample the next day, or just left in the box until imaging.

FRET

FRET Protocol for Negative Controls

Part 1

Dilute mCerulean3 (Cerulean) and mVenus (Venus) protein samples to 1 mg/mL (≈ 34 µM) with PBS pH 7.4
Create a serial dilution of both Cerulean and Venus from 1 mg/mL down to 1 µg/mL in an (ideally) black sided 96-well plate (200 µL of undiluted protein, transfer 20 µL to 180 µL PBS, and so on).
Scan the excitation and emission spectra of these wells to determine the ideal values of excitation and emission for each value, using the values from Markwardt et al. and Jonáš et al. as shown in the table as a starting point.
Blank appropriately with 180 µL PBS.

	Excitation	Emission
mCerulean3	433 (400-465)	475 (465-525)
mVenus	515 (500-525)	528 (520-550)

The values in these tables were taken from, Markwardt et al. (2011)¹ and Jonáš et al. (2014)².

Dilute samples equally if concentration is too high for the fluorescence reader of the machine.

Part 2

Add 90 µL of 1 mg/mL protein to 90 µL PBS for each protein in triplicate.
Add 90 µL of Cerulean to 90 µL Venus in triplicate.
Blank with 180 µL PBS.
Excite all wells at the ideal excitation value determined for Cerulean and scan wavelengths longer than this.
Please see supplementary data for specific parameters used.

Enzyme Assays

BCA Protein Assay

Protocol followed as per Pierce BCA Protein Assay Kit 23225 to determine protein concentration in solution:

Sample to working reagent ratio = 1:8
96-well plate reader: SPECTROstar Nano BMG LABTECH
Software used: SPECTROstar Nano

Salkowski Assay

The Salkowski assay is a measurement-based protocol used to assess the quantity of indole-3-acetic acid within a sample. This particular protocol has been adapted from the methods used by 2011 iGEM Imperial College London, Tang and Bonner’s 1947 paper³, and Glickmann and Dessaux's 1995 paper⁴.

Cellular Protocol

Pick a single colony of untransformed T7 and transformed T7 and inoculate a 2-5ml overnight culture in LB.
Add antibiotics to the transformed culture.
The next day, add 50 mL of LB + 2.5 mg/mL tryptophan to a 250 mL shake flask labelled “Control”.
Add 50 mL of LB + 2.5 mg/mL tryptophan + antibiotic to another baffled shake flask labelled “IAA”.
Add 1 mL of the respective overnight cultures to each flask. Incubate with shaking (200 rpm) at 37°C. Sample at 1h, 4h, 7h and 20-24h.
For each sample: a. Measure the OD at 600 nm.
Spin down 1 mL. Retain pellet and freeze. Retain supernatant and store at 4°C. Label these 4.
The next day: Add Salkowski reagent to supernatants at the ratio of 1 mL per 0.5 mL of supernatant. Incubate in the dark for 30 minutes.
Add 1 mL of Salkowski reagent to each cell pellet.
Vortex to mix.
Incubate in the dark for 30 minutes.
Centrifuge at full speed for 5 minutes.
Measure the absorbance at 530 nm Measure appropriate controls as well.
Normalise readings at each time point against OD₆₀₀.

The assay is performed by adding the Salkowski reagent to a prepared sample:

To prepare the Salkowski assay reagent, slowly add 95 % H₂SO₄ to Milli-Q water in a ratio of 3:2, allowing time for heat dissipation between additions.
Dissolve 4.5 g/L of anhydrous FeCl₃ into the solution. Store in the dark, wrapped in foil.
Prepare the standard by using a 3 mM stock, then diluted 1 in 10 for usage – with 300 µM to 0 µM concentrations.
Prepare a standard curve.
Samples are run by adding the sample (or standard) to the Salkowski reagent in a ratio of 1:2 in a time sensitive manner within a 96-well microtitre plate.
Run the plate on a plate reader for absorbance at 530 nm, after being left for 30 minutes in the dark.

High Performance Liquid Chromotography (HPLC)

Acidify supernatant with 5 M HCl to pH 2.5.
Extract supernatant with an equal volume of ethyl acetate.
Evaporate off the solvent by either drying it through a vacuum or dissolving in methanol & acetic acid at pH 4.5 (25 % methanol, 1 % acetic acid, made up to 1 mL with H₂O).
250 µL methanol, 10 µL acetic acid, 740 µL H₂O.
Run system with solution 72 % of 1 % acetic acid and 28 % of 100 % methanol for 0.8 mL/minute with injection of 30.

Plants

Seed Surface Sterilisation

Wild-type Arabidopsis thaliana seeds were surface sterilised by mixing in a solution of 2.5 % sodium hypochlorite with a drop of Tween for 10 minutes.
Seeds were subsequently washed eight times in sterile water.

Indole-3-acetic Acid Filter Sterilisation

10 mM stock indole-3-acetic acid (IAA) was prepared by dissolving indole-3-acetic acid sodium salt purchased from Sigma Aldrich in ethanol.
In a laminar flow hood, the solution was filter sterilised through a 0.22 µm Millex® syringe driven filter and stored at 4°C in a foil covered container to protect from light.

MS Agar Plate Preparation

Murashige and Skoog (MS) media was prepared using M5519 MS basal powdered medium purchased from Sigma Aldrich.
In four separate Schott bottles, 250 mL of MS media was prepared using 1.1 g of MS basal salts (per manufacturer instructions) and Milli-Q purified water.
Each mixture was supplemented with sucrose (1 % w/v) and agar powder (0.8 % w/v) and adjusted to pH 5.7 using KOH.
Mixtures were autoclaved and allowed to cool to approximately 50°C (or until able to be held comfortably).
Cooled bottles were transferred to a dark laminar flow hood where indole-3-aecetic acid solution was added to prepare four separate MS agar mixtures at 0 µM, 1 µM, 10 µM and 100 µM concentrations IAA respectively.
In a dark laminar flow hood, 50 mL of media was pipetted into a square plate using a serological pipette. Four 50 mL plates were prepared for each concentration of IAA.
Plates were labelled, sealed with parafilm and stored wrapped in foil at 4°C until further use.

Liquid Culture Media Preparation

MS media was prepared using M5519 MS basal powdered medium purchased from Sigma Aldrich.
In an autoclavable Schott bottle, 500 mL 1/2 MS media was prepared using 1.1 g of MS basal salts and 500 ml of Milli-Q purified water (4.4 g salts equivalent to 1 L MS media per manufacturer’s instructions).
Media was supplemented with sucrose (1 % w/v) and adjusted to pH 5.7 using KOH.
Media was autoclaved and stored at room temperature until further use.

Arabidopsis thaliana Growth on Agar Plates

Surface sterilised seeds were added to a flask containing an autoclaved solution of Milli-Q water with 1 % w/v sucrose in a laminar flow hood.
This flask was covered with foil and seeds were stratified at 4°C for two days.
Stratified seeds were then allowed to germinate under light at room temperature for 2 days.
In a dark laminar flow hood, seedlings were transplanted to MS media plates with varying concentrations of IAA. Approximately five seedlings were transplanted per plate using sterile tweezers and loops. Seedlings were arranged in a line, 2 cm from the top of each plate. Three plates with seedlings were prepared for each concentration.
Plates were sealed with parafilm and covered with foil leaving only the top 2 cm exposed in order to limit IAA photodegradation and simulate the natural growth conditions of higher plants whereby only shoots are exposed to illumination⁵. Plates were stored upright at an approximately 90° angle. The plated seedlings were grown under light at room temperature for 20 days.
Primary root length and lateral root numbers were measured at 4, 6, 11 and 20 days following the initial seedling transplant.

Arabidopsis thaliana Liquid Culture

In a laminar flow hood, 25 mL of 1/2 MS media with 1 % sucrose was aliquoted into twelve 50 mL foil wrapped falcon tubes.
Filter sterilised IAA was added to prepare duplicate tubes at 0 nM, 1nM, 10 nM, 100 nM, 1 µM and 10 µM concentrations of IAA.
100 µL of surface sterilised A. thaliana seeds were added to each tube.
Liquid cultures were stratified at 4°C for two days, then grown under light at room temperature for 9 days.
Three seedlings from each tube were selected at random and measured for primary root length and shoot length.

References

Markwardt, M. et al. An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching. PLOS ONE 6 e17896 (2011).
Jonáš, A. et al. In vitro and in vivo biolasing of fluorescent proteins suspended in liquid microdroplet cavities. Lab Chip 14 3093-3100 (2014).
Tang, YW. and Bonner, J. The enzymatic inactivation of indoleacetic acid; some characteristics of the enzyme contained in pea seedlings. Arch. Biochem. 13 11–25 (1947).
Glickmann, E. and Dessaux, Y. A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Applied and Environmental Microbiology 61 793–796 (1995).
Xu, W. et al. An improved agar-plate method for studying root growth and response of Arabidopsis thaliana. Sci Rep 3 1273 (2013).

@@ Line 2: / Line 2: @@
 {{Template:UNSW_Australia/Header}}
 {{Template:UNSW_Australia/Basics}}
+{{Template:UNSW_Australia/Up}}
 <html>
@@ Line 22: / Line 23: @@
 <div id=experiments-content class="to-load">
 <br/>
-<p>This page includes all the experimental protocols used by the UNSW iGEM team. Please click on each heading to view the protocols or view our weekly progress on the <a href=http://https://2018.igem.org/Team:UNSW_Australia/Notebook>notebook</a> page. Alternatively, to see the detailed experimental introduction, results and discussion, please visit the <a href=https://2018.igem.org/Team:UNSW_Australia/Lab>lab overview</a> page.</p>
+<p>This page includes all the experimental protocols used by the UNSW iGEM team. Please click on each heading to view the protocols or view our weekly progress on the <a href=https://2018.igem.org/Team:UNSW_Australia/Notebook>notebook</a> page. Alternatively, to see the detailed experimental introduction, results and discussion, please visit the <a href=https://2018.igem.org/Team:UNSW_Australia/Lab>lab overview</a> page.</p>
 <br/>
@@ Line 34: / Line 35: @@
 <ol>
 <li>Forward and reverse primers created for the plasmids.</li>
-<li>2 &micro;l of plasmid + 198 &micro;l of water.</li>
+<li>2 &micro;L of plasmid + 198 &micro;L of water.</li>
 <li>PCR amplification, according to the table below:</li>
 </ol>
@@ Line 42: / Line 43: @@
 <th>Component</th>
 </td>
-<th>50 &micro;l Reaction</th>
+<th>50 &micro;L Reaction</th>
 </td>
 <th>Final Concentration</th>
@@ Line 54: / Line 55: @@
 </td>
 <td
-<p>25 &micro;l</p>
+<p>25 &micro;L</p>
 </td>
 <td
@@ Line 65: / Line 66: @@
 </td>
 <td>
-<p>2.5 &micro;l</p>
+<p>2.5 &micro;L</p>
 </td>
 <td>
@@ Line 76: / Line 77: @@
 </td>
 <td>
-<p>2.5 &micro;l</p>
+<p>2.5 &micro;L</p>
 </td>
 <td>
@@ Line 146: / Line 147: @@
 </td>
 <td>
-<p>2 min</p>
+<p>2 minutes</p>
 </td>
 </tr>
@@ Line 184: / Line 185: @@
 </td>
 <td>
-<p>1 &micro;l</p>
+<p>1 &micro;L</p>
 </td>
 </tr>
@@ Line 200: / Line 201: @@
 </td>
 <td>
-<p>5 &micro;l (1X)</p>
+<p>5 &micro;L (1X)</p>
 </td>
 </tr>
@@ Line 208: / Line 209: @@
 </td>
 <td>
-<p>50 &micro;l</p>
+<p>50 &micro;L</p>
 </td>
 </tr>
@@ Line 222: / Line 223: @@
 <ol>
 <li>Combine 1 g of agarose powder with 100 mL of 1x TAE buffer in a microwavable flask.</li>
-<li>Microwave for 1-2 min until the agarose is completely dissolved. Avoid over-boiling, and stop to swirl the flask every 20-30 seconds until the solution is clear.</li>
+<li>Microwave for 1-2 minutes until the agarose is completely dissolved. Avoid over-boiling, and stop to swirl the flask every 20-30 seconds until the solution is clear.</li>
 <li>Allow the solution to cool down until you can comfortably hold the flask with your hand, then add 1 &micro;L of RedSafe.</li>
-<li>Seal the ends of a gel tray and pour the solution into the tray with a well comb in place. Let the solution sit at room temperature for 20-30 mins until it solidifies into a gel.</li>
+<li>Seal the ends of a gel tray and pour the solution into the tray with a well comb in place. Let the solution sit at room temperature for 20-30 minutes until it solidifies into a gel.</li>
 <li>Place the gel into the gel tank, and fill the tank with 1X TAE buffer until the gel is covered. Remove the well comb.</li>
 <li>Mix 2 &micro;L of the DNA sample with 3 &micro;L of H<sub>2</sub>O and 1 &micro;L of 6X loading dye.</li>
@@ Line 245: / Line 246: @@
 <tr>
 <td>1 M MgCl<sub>2</sub></td>
-<td>300 μL</td>
+<td>300 &micro;L</td>
 </tr>
 <tr>
 <td>100 mM dGTP</td>
-<td>60 μL</td>
+<td>60 &micro;L</td>
 </tr>
 <tr>
 <td>100 mM dATP</td>
-<td>60 μL</td>
+<td>60 &micro;L</td>
 </tr>
 <tr>
 <td>100 mM dTTP</td>
-<td>60 μL</td>
+<td>60 &micro;L</td>
 </tr>
 <tr>
 <td>100 mM dCTP</td>
-<td>60 μL</td>
+<td>60 &micro;L</td>
 </tr>
 <tr>
 <td>1 M DTT</td>
-<td>300 μL</td>
+<td>300 &micro;L</td>
 </tr>
 <tr>
@@ Line 273: / Line 274: @@
 <tr>
 <td>100 mM NAD</td>
-<td>300 μL</td>
+<td>300 &micro;L</td>
 </tr>
 <tr>
 <td>H<sub>2</sub>O</td>
-<td>360 μL</td>
+<td>360 &micro;L</td>
 </tr>
 </tbody>
 </table>
 <br/>
-<p>Assembly Master Mix (1.2 mL total, store in 15 µL aliquots at -20&deg;C.):</p>
+<p>Assembly Master Mix (1.2 mL total, store in 15 &micro;L aliquots at -20&deg;C.):</p>
 <table class="lab-table">
 <tbody>
 <tr>
 <td>5X Isothermal Master Mix</td>
-<td>320 μL</td>
+<td>320 &micro;L</td>
 </tr>
 <tr>
-<td>510 U/μL T5 exonuclease</td>
+<td>510 U/&micro;L T5 exonuclease</td>
-<td>0.64 μL</td>
+<td>0.64 &micro;L</td>
 </tr>
 <tr>
-<td>2 U/μL Phusion DNA Polymerase</td>
+<td>2 U/&micro;L Phusion DNA Polymerase</td>
-<td>20 μL</td>
+<td>20 &micro;L</td>
 </tr>
 <tr>
-<td>40 U/μL T4 DNA Ligase</td>
+<td>40 U/&micro;L T4 DNA Ligase</td>
-<td>0.16 μL</td>
+<td>0.16 &micro;L</td>
 </tr>
 <tr>
 <td>H<sub>2</sub>O</td>
-<td>860 μL</td>
+<td>860 &micro;L</td>
 </tr>
 </tbody>
@@ Line 312: / Line 313: @@
 <li>PCR amplify or digest your fragments of choice and gel purify.</li>
 <li>If PCR from a methylated DNA template (e.g. linearised plasmid), a DpnI digest can be used to remove the unwanted template plasmid. Clean up afterwards.</li>
-<li>Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.</li>
+<li>Thaw a 15 &micro;L assembly mixture aliquot and keep on ice until ready to be used.</li>
-<li>Nanodrop your DNA fragments. Prepare 50-100 ng of vector with 2-3 fold excess of insert in nuclease-free water to a total volume of 5 μL.</li>
+<li>Nanodrop your DNA fragments. Prepare 50-100 ng of vector with 2-3 fold excess of insert in nuclease-free water to a total volume of 5 &micro;L.</li>
-<li>Add 15 μl assembly mixture to the DNA mixture, for a total reaction volume of 20 μL. </li>
+<li>Add 15 &micro;L assembly mixture to the DNA mixture, for a total reaction volume of 20 &micro;L. </li>
-<li>Incubate at 50&deg;C for 15 to 60 min (60 min is optimal).</li>
+<li>Incubate at 50&deg;C for 15 to 60 minutes (60 minutes is optimal).</li>
 </ol>
 </div>
@@ Line 322: / Line 323: @@
 <h2>Heat Shock Transformation</h2>
 <ol>
-<li>Incubate 50 ng of plasmid construct  with 25 µL of chemically competent <i>E. coli</i> T7 express or DH5&alpha; on ice for 30 minutes.</li>
+<li>Incubate 50 ng of plasmid construct  with 25 &micro;L of chemically competent <i>E. coli</i> T7 express or DH5&alpha; on ice for 30 minutes.</li>
 <li>Heat shock the cells for 45 seconds at 42&deg;C and place back onto ice for 2 minutes.</li>
-<li>Allow cells to grow for 45 minutes in 200 µL of SOC outgrowth media (NEB) at 37&deg;C and 200 rpm.</li>
+<li>Allow cells to grow for 45 minutes in 200 &micro;L of SOC outgrowth media (NEB) at 37&deg;C and 200 rpm.</li>
 <li>Spread plate onto Luria Broth (LB) agar plates containing appropriate antibiotics at working concentration and grow at 37&deg;C overnight.</li>
 </ol>
@@ Line 332: / Line 333: @@
 <h2>Colony PCR</h2>
 <ol>
-<li>Pick up individual bacterial colonies from an agar plate that was grown overnight using a pipette tip, and dilute each colony into 50 μL of water.</li>
+<li>Pick up individual bacterial colonies from an agar plate that was grown overnight using a pipette tip, and dilute each colony into 50 &micro;L of water.</li>
 <li>Prepare the PCR master mix by multiplying the volume for each component shown below (for 1 reaction) by the number of colonies to sample +1 (i.e. number of reactions plus 1 excess).
 <ul>
-<li>18 uL nuclease free water</li>
+<li>18 &micro;L nuclease free water.</li>
-<li>5 uL 5x Taq master mix</li>
+<li>5 &micro;L 5X Taq master mix.</li>
-<li>0.5 uL 10 uM T7 promoter primer</li>
+<li>0.5 &micro;L 10 &micro;M T7 promotor primer</li>
-<li>0.5 uL 10 uM T7 terminator primer</li>
+<li>0.5 &micro;L 10 &micro;M T7 terminator primer</li>
 </ul>
 </li>
-<li>Add 1 uL of each diluted colony and 24 uL of PCR master mix to a PCR tube.</li>
+<li>Add 1 &micro;L of each diluted colony and 24 &micro;L of PCR master mix to a PCR tube.</li>
-<li>Run PCR with the following steps (lid at 105&deg;C and volume = 20 uL):
+<li>Run PCR with the following steps (lid at 105&deg;C and volume = 20 &micro;L):
 <ul>
-<li>Step 1: 95&deg;C for 5 mins</li>
+<li>Step 1: 95&deg;C for 5 minutes</li>
-<li>Step 2: 95&deg;C for 30 secs</li>
+<li>Step 2: 95&deg;C for 30 seconds</li>
-<li>Step 3: 55&deg;C for 30 secs</li>
+<li>Step 3: 55&deg;C for 30 seconds</li>
-<li>Step 4: 68&deg;C for 2 mins</li>
+<li>Step 4: 68&deg;C for 2 minutess</li>
-<li>25-35 cycles of steps 2-4</li>
+<li>Repeat 25-35 cycles of steps 2-4</li>
-<li>Step 5: 68&deg;C 5 mins</li>
+<li>Step 5: 68&deg;C 5 minutes</li>
-<li>Then hold at 4&deg;C</li>
+<li>Step 6: Hold at 4&deg;C</li>
 </ul></li>
-<li>Run PCR products on a 1% agarose gel and image for analysis.</li>
+<li>Run PCR products on a 1 % agarose gel and image for analysis.</li>
 </ol>
 </div>
@@ Line 359: / Line 360: @@
 <h2>Sequencing</h2>
 <ol>
-<li>Transfer 10 uL of purified plasmid sample (50-100ng/uL) to a PCR tube. Add 5 uL of one primer (forward or reverse).</li>
+<li>Transfer 10 &micro;L of purified plasmid sample (50-100 ng/&micro;L) to a PCR tube. Add 5 &micro;L of one primer (forward or reverse).</li>
 <li>Request sequencing at the UNSW Centre for Genomics via the on-line portal.</li>
 <li>Label PCR tubes carefully with numbers as per the on-line order.</li>
@@ Line 368: / Line 369: @@
 <br/>
 <div id=restriction class=cloning protocol>
-<h2>Restriction cloning</h2>
+<h2>Restriction Cloning</h2>
 <ol>
 <li>Set-up the reaction mixture:
@@ Line 378: / Line 379: @@
 </td>
 <td>
-<p>1 &micro;l of each enzyme</p>
+<p>1 &micro;L of each enzyme</p>
 </td>
 </tr>
@@ Line 394: / Line 395: @@
 </td>
 <td>
-<p>5 &micro;l (1X)</p>
+<p>5 &micro;L (1X)</p>
 </td>
 </tr>
@@ Line 402: / Line 403: @@
 </td>
 <td>
-<p>50 &micro;l</p>
+<p>50 &micro;L</p>
 </td>
 </tr>
@@ Line 408: / Line 409: @@
 </table>
 </li>
-<li>Incubate for 1 hr at 37&deg;C</li>
+<li>Incubate for 1 hr at 37&deg;C.</li>
-<li>Heat inactivate at 80&deg;C for 20 minutes</li>
+<li>Heat inactivate at 80&deg;C for 20 minutes.</li>
 </ol>
 <h3>Ligation</h3>
@@ Line 418: / Line 419: @@
 <tr>
 <th>Component</th>
-<th>20 μl Reaction</th>
+<th>20 &micro;L Reaction</th>
 </tr>
 <tr>
@@ Line 425: / Line 426: @@
 </td>
 <td>
-<p>2 μl</p>
+<p>2 &micro;L</p>
 </td>
 </tr>
@@ Line 449: / Line 450: @@
 </td>
 <td>
-<p>to 20 μl</p>
+<p>to 20 &micro;L</p>
 </td>
 </tr>
@@ Line 457: / Line 458: @@
 </td>
 <td>
-<p>1 μl</p>
+<p>1 &micro;L</p>
 </td>
 </tr>
@@ Line 465: / Line 466: @@
 <li>For cohesive (sticky) ends, incubate at 16&deg;C overnight or room temperature for 10 minutes.</li>
 <li>Heat inactivate at 65&deg;C for 10 minutes.</li>
-<li>Chill on ice and transform 1-5 μl of the reaction into 25 μl competent cells.</li>
+<li>Chill on ice and transform 1-5 &micro;L of the reaction into 25 &micro;L competent cells.</li>
 </ol>
 </div>
@@ Line 482: / Line 483: @@
 <div id=protein class="toggle-content">
          <div id=starter-culture class=protocol protein>
-             <h2>Starter culture</h2>
+             <h2>Starter Culture</h2>
-                 <p>One colony was selected from the plate grown overnight and grown in 2 mL of LB containing appropriate antibiotic at 37°C and 200 rpm overnight.</p>
+                 <p>One colony was selected from the plate grown overnight and grown in 2 mL of LB containing appropriate antibiotic at 37&deg;C and 200 rpm overnight.</p>
          </div>
          <div id=grow-up class=protocol protein>
              <h2>Large-scale grow-up</h2>
                  <ol>
-                     <li>Baffled shake flasks containing 500 mL of LB with 50uL of the appropriate antibiotic at 37°C are inoculated with the starter culture.</li>
+                     <li>Baffled shake flasks containing 500 mL of LB with 50uL of the appropriate antibiotic at 37&deg;C are inoculated with the starter culture.</li>
-                     <li>The cells are grown at 37°C and 200 rpm and OD600 is periodically measured. </li>
+                     <li>The cells are grown at 37&deg;C and 200 rpm and OD<sub>600</sub> is periodically measured. </li>
-                     <li>Once OD600 reaches above 0.6, add 1mM of IPTG to induce expression of the proteins.</li>
+                     <li>Once OD<sub>600</sub> reaches an absorbance reading of 0.6, add 1mM of IPTG to induce expression of the protein.</li>
-                     <li>After induction, grow the cells overnight at 24°C, 200 rpm.</li>
+                     <li>After induction, grow the cells overnight at 24&deg;C, 200 rpm.</li>
                  </ol>
          </div>
          <div id=cell-collection class=protocol protein>
-             <h2>Collection of cells by centrifugation</h2>
+             <h2>Collection of Cells by Centrifugation</h2>
                  <ol>
                      <li>Centrifuge the bacterial culture at 4600 x g for 20 minutes.</li>
-                     <li>Collect cell pellet and resuspended in binding buffer (20mM NaH2PO4, 500mM NaCl, 10mM Imidazole).</li>
+                     <li>Collect cell pellet and resuspended in binding buffer (20 mM NaH<sub>2</sub>PO<sub>4</sub>, 500 mM NaCl, 10 mM Imidazole).</li>
                  </ol>
          </div>
          <div id=lysis class=protocol protein>
-             <h2>Cell lysis by sonication</h2>
+             <h2>Cell Lysis by Sonication</h2>
                  <ol>
-                     <li>Lyse the cell pellet by sonication (Branson) for 10 minutes at 50% amplitude at alternating 2 second intervals, kept on ice.</li>
+                     <li>Lyse the cell pellet by sonication (Branson) for 10 minutes at 50% amplitude, alternating 2 second intervals, kept on ice.</li>
                      <li>Centrifuge the cell lysate at 15000 rpm for 45 minutes.</li>
                      <li>Collect the supernatant (soluble fraction).</li>
@@ Line 511: / Line 512: @@
          <div id=IMAC class=protocol protein>
              <h2>IMAC</h2>
-                 <p>Immobilised metal ion affinity chromatography (IMAC) was performed to purify the expressed proteins.</p>
+                 <p>Immobilised Metal ion Affinity Chromatography (IMAC) was performed to purify the expressed proteins.</p>
                  <ol>
                      <li>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.</li>
-                     <li>Wash with 10 mL of binding buffer (20mm NaH2PO4, 500mM NaCl, 10mM Imidazole).</li>
+                     <li>Wash with 10 mL of binding buffer (20 mM NaH<sub>2</sub>PO<sub>4</sub>, 500 mM NaCl, 10 mM Imidazole).</li>
-                     <li>Elute with 2 mL of elution buffer (same as binding buffer, but with 500 mM Imidazole).</li>
+                     <li>Elute with 2 mL of elution buffer (20 mM NaH<sub>2</sub>PO<sub>4</sub>, 500 mM NaCl, 500 mM Imidazole).</li>
-                     <li>Collect fractions with SDS-PAGE.</li>
+                     <li>Analyse fractions with SDS-PAGE.</li>
                  </ol>
          </div>
          <div id=buffer-exchange class=protocol protein>
-             <h2>Buffer exchange</h2>
+             <h2>Buffer Exchange</h2>
                  <h3>Column</h3>
                  <ol>
                      <li>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).</li>
-                     <li>Add protein to the column</li>
+                     <li>Add protein to the column.</li>
-                     <li>Top up column with PBS buffer</li>
+                     <li>Top up column to 6 mL with PBS buffer.</li>
                      <li>Centrifuge column at 4600 x g for 20 minutes.</li>
-                     <li>Repeatedly centrifuge, discard flow through, and top up with PBS buffer (pH 8) until dilution factor of 0.1 is reached. That is, until there is 1% of the old buffer left in the solution.</li>
+                     <li>Repeatedly centrifuge, discard flow through, and top up with PBS buffer (pH 8) until dilution factor of 0.01 is reached. That is, until there is 1% of the old buffer left in the solution.</li>
                  </ol>
                  <h3>Dialysis</h3>
                  <ol>
-                     <li>Add 1mL of protein and 1mL of PBS buffer (pH 8) to a 15mL Falcon tube.</li>
+                     <li>Add 1 mL of protein and 1 mL of PBS buffer (pH 8) to a 15 mL Falcon tube.</li>
-                     <li>Add 2mL of the solution to a SnakeSkin™ Dialysis Tubing, 10K MWCO, 22 mm.</li>
+                     <li>Add 2 mL of the solution to a SnakeSkin™ Dialysis Tubing, 10K MWCO, 22 mm.</li>
-                     <li>Use dialysis tubing clamps (one-piece polypropylene clamp) to further secure the solution inside the snakeskin dialysis tubing.</li>
+                     <li>Use dialysis tubing clamps (one-piece polypropylene clamp) to further secure the solution inside the SnakeSkin dialysis tubing.</li>
-                     <li>Add 500mL of PBS buffer, pH 8, (this is the buffer we want to exchange into) into a 500ml glass beaker.</li>
+                     <li>Add 500 mL of PBS buffer, pH 8, (this is the buffer we want to exchange into) into a 500 mL glass beaker.</li>
                      <li>Place the dialysis tubing with the solution into the beaker.</li>
                      <li>Place the beaker on top of a magnetic stirrer, 75 rpm, and leave overnight.</li>
@@ Line 547: / Line 548: @@
                          <li>Mini iBlotTM stack</li>
                          <li>TBS-T: </li>
-                         <li>1x TBS with 0.1% Tween20</li>
+                         <li>1X TBS with 0.1 % Tween20</li>
                          <li>Blocking Solution: </li>
-                         <li>5% skim milk in TBS-T</li>
+                         <li>5 % skim milk in TBS-T</li>
                          <li>Antibody Solution: </li>
-                         <li>1:2000 dilution of HRP conjugated anti-His-tag antibody in TBS-T + 1% BSA</li>
+                         <li>1:2000 dilution of HRP conjugated anti-His-tag antibody in TBS-T + 1 % BSA</li>
                          <li>Chemiluminescent HRP substrate</li>
                      </ul>
                  <h3>Sample Preparation</h3>
                      <ol>
-                         <li>Add reducing buffer to the bacterial lysates</li>
+                         <li>Add reducing buffer to the bacterial lysates.</li>
-                         <li>Heat at 95 degrees for 5 minutes</li>
+                         <li>Heat at 95&deg;C for 5 minutes.</li>
                      </ol>
-                 <h3>SDA-Page Gel</h3>
+                 <h3>SDS-Page Gel</h3>
                      <ol>
-                         <li>Remove the NuPAGE gel from its packaging and peel off the plastic strip from its base</li>
+                         <li>Remove the NuPAGE gel from its packaging and peel off the plastic strip from its base.</li>
-                         <li>Place the gel inside the tank, and fill with NuPAGE MES running buffer</li>
+                         <li>Place the gel inside the tank, and fill with NuPAGE MES running buffer.</li>
-                         <li>Load 5uL of the protein standards ladder into the first well</li>
+                         <li>Load 5 &micro;L of the protein standards ladder into the first well.</li>
-                         <li>Load up to 20uL of each lysate sample into the wells</li>
+                         <li>Load up to 20 &micro;L of each lysate sample into the wells.</li>
-                         <li>Connect the gel tank to a power pack, and run at 160V for 40min</li>
+                         <li>Connect the gel tank to a power pack, and run at 160 V for 40 minutes.</li>
                      </ol>
                  <h3>Protein Transfer</h3>
                      <ol>
-                         <li>Remove and rinse the gel in water</li>
+                         <li>Remove and rinse the gel in water.</li>
-                         <li>Inside an iBlot Transfer Device, assemble the mini stack with the gel inside</li>
+                         <li>Inside an iBlot Transfer Device, assemble the mini stack with the gel inside.</li>
-                         <li>Run at 20V for 7min</li>
+                         <li>Run at 20 V for 7 minutes.</li>
                      </ol>
                  <h3>Blocking</h3>
-                     <ol><li>Incubate the membrane for 1-2hrs in blocking solution at room temperature, shaking</li></ol>
+                     <ol><li>Incubate the membrane for 1-2 hours in blocking solution at room temperature, shaking.</li></ol>
                  <h3>Antibody Staining</h3>
                      <ol>
-                         <li>Incubate the membrane in antibody solution either at 4 degrees overnight, or at room temperature for 2 hours</li>
+                         <li>Incubate the membrane in antibody solution either at 4&deg;C overnight, or at room temperature for 2 hours.</li>
-                         <li>Wash the membrane in TBS-T three times for 10mins per wash at room temperature, shaking</li>
+                         <li>Wash the membrane in TBS-T three times for 10 minutes per wash at room temperature, shaking.</li>
                      </ol>
                  <h3>Detection</h3>
                      <ol>
-                         <li>Remove membrane from the last wash and place in chemiluminescent image analyser</li>
+                         <li>Remove membrane from the last wash and place in chemiluminescent image analyser.</li>
-                         <li>Prepare HRP substrate according to manufacturer's instructions and add to the membrane</li>
+                         <li>Prepare HRP substrate according to manufacturer's instructions and add to the membrane.</li>
-                         <li>Image</li>
+                         <li>Image.</li>
                      </ol>
          </div>
@@ Line 598: / Line 599: @@
              <h2>Catcher and Tag Assembly</h2>
              <ol>
-                 <li>Purified IaaH fused to SpyTag (IaaH-SpyTag) and prefoldin proteins fused to SpyCatcher (aPFD-SpyCatcher, gPFD-SpyCatcher and SpyCatcher-gPFD-SpyCatcher) were obtained </li>
+                 <li>Purified IaaH fused to SpyTag (IaaH-SpyTag) and prefoldin proteins fused to SpyCatcher (aPFD-SpyCatcher, gPFD-SpyCatcher and SpyCatcher-gPFD-SpyCatcher) were obtained.</li>
-                 <li>IaaH-SpyTag was mixed with either aPFD-SpyCatcher, gPFD-SpyCatcher and SpyCatcher-gPFD-SpyCatcher at a concentration of 3 µM and 15 µM respectively in a total volume of 250 µL in PBS pH 8, and incubated at room temperature. </li>
+                 <li>IaaH-SpyTag was mixed with either aPFD-SpyCatcher, gPFD-SpyCatcher and SpyCatcher-gPFD-SpyCatcher at a concentration of 3 &micro;M and 15 &micro;M respectively in a total volume of 250 &micro;L in PBS pH 8, and incubated at room temperature.</li>
-                 <li>After 0, 10, 20 and 30 minutes of incubation, a 10 µL sample was obtained.</li>
+                 <li>After 0, 10, 20 and 30 minutes of incubation, a 10 &micro;L sample was obtained.</li>
-                 <li>5 µL of 4x Bolt LDS sample buffer was added to each sample, and then boiled for 10 minutes at 95&deg;C to cease SpyCatcher reactivity whilst preserving any covalent interactions.</li>
+                 <li>5 &micro;L of 4X Bolt LDS sample buffer was added to each sample, and then boiled for 10 minutes at 95&deg;C to cease SpyCatcher reactivity whilst preserving any covalent interactions.</li>
                  <li>The samples were then examined via SDS-PAGE.</li>
              </ol>
@@ Line 611: / Line 612: @@
          <div id=TEM class=protocol assembly>
              <h2>TEM</h2>
-                  <p>Materials</p>
+                  <h3>Materials</h3>
-<ol>
+<ul>
 <li>Protein sample (ideally in water or low salt buffer)</li>
-<li>MilliQ water</li>
+<li>Milli-Q water</li>
-<li>2% uranyl acetate</li>
+<li>2 % uranyl acetate</li>
 <li>Parafilm</li>
 <li>Self-closing forceps</li>
-</ol>
+</ul>
-<p>Staining</p>
+<h3>Staining</h3>
 <ol>
-<li>Glow discharge the carbon-coated grids (carbon-coated side up) to render them hydrophilic</li>
+<li>Glow discharge the carbon-coated grids (carbon-coated side up) to render them hydrophilic.</li>
-<li>Place the grids (carbon-coated side up) on parafilm</li>
+<li>Place the grids (carbon-coated side up) on parafilm.</li>
-<li>Place 7 µL of sample on a grid</li>
+<li>Place 7 &micro;L of sample on a grid.</li>
-<li>Let the grid stain for 10 minutes</li>
+<li>Let the grid stain for 10 minutes.</li>
-<li>Using a pasteur pipette, transfer 5 droplets of MilliQ water on a piece parafilm (for each grid)</li>
+<li>Using a pasteur pipette, transfer 5 droplets of Milli-Q water on a piece parafilm (for each grid).</li>
 <li>After the 10 minutes of staining, pick up grid with the forceps and wash residue salts by passing the grid through the five droplets of water.</li>
-<li>In a fume hood, place one droplet of 2% uranyl acetate on a piece of parafilm (for each grid)</li>
+<li>In a fume hood, place one droplet of 2 % uranyl acetate on a piece of parafilm (for each grid).</li>
 <li>Wear appropriate PPE to handle uranium; do not work with uranium outside the fume hood.</li>
-<li>Place the grid on the uranyl acetate drop (carbon-coated side down) and stain for 5-7minutes.</li>
+<li>Place the grid on the uranyl acetate drop (carbon-coated side down) and stain for 5-7 minutes.</li>
-<li>Pick the grid up with the forceps</li>
+<li>Pick the grid up with the forceps.</li>
-<li>Remove excess uranyl acetate by touching one side of the grid with filter paper for a few seconds, then briefly touch the other side of the grid with the filter paper</li>
+<li>Remove excess uranyl acetate by touching one side of the grid with filter paper for a few seconds, then briefly touch the other side of the grid with the filter paper.</li>
-<li>Let the grid dry for a few minutes before transferring back it to its box</li>
+<li>Let the grid dry for a few minutes before transferring back it to its box.</li>
-<li>The grid can be fully dried in a oven if imaging the sample the next day, or just left in the box until imaging</li>
+<li>The grid can be fully dried in a oven if imaging the sample the next day, or just left in the box until imaging.</li>
 </ol>
          </div>
@@ Line 646: / Line 647: @@
              <h2>FRET Protocol for Negative Controls</h2>
                  <h3>Part 1</h3>
-                 <ul>
+                 <ol>
-                     <li>Dilute mCerulean3 (Cerulean) and mVenus (Venus) protein samples to 1 mg/mL (&asymp; 34 uM) with PBS pH 7.4</li>
+                     <li>Dilute mCerulean3 (Cerulean) and mVenus (Venus) protein samples to 1 mg/mL (&asymp; 34 &micro;M) with PBS pH 7.4</li>
-                     <li>Create a serial dilution of both Cerulean and Venus from 1mg/mL down to 1ug/mL in an (ideally) black sided 96 well plate (200 uL of undiluted protein, transfer 20 uL to 180 uL PBS, and so on).</li>
+                     <li>Create a serial dilution of both Cerulean and Venus from 1 mg/mL down to 1 &micro;g/mL in an (ideally) black sided 96-well plate (200 &micro;L of undiluted protein, transfer 20 &micro;L to 180 &micro;L PBS, and so on).</li>
-                     <li>Scan the excitation and emission spectra of these wells to determine the ideal values of excitation and emission for each value, using the values from Markwardt et al. and Jonáš et al. as shown in the table as a starting point.</li>
+                     <li>Scan the excitation and emission spectra of these wells to determine the ideal values of excitation and emission for each value, using the values from Markwardt <i>et al.</i> and Jon&aacute;&scaron; <i>et al.</i> as shown in the table as a starting point.</li>
-                     <li>Blank appropriately with 180 uL PBS.</li>
+                     <li>Blank appropriately with 180 &micro;L PBS.</li>
-                 </ul>
+                 </ol>
                  <br/>
                      <table class="lab-table">
@@ Line 671: / Line 672: @@
                      </table>
                  <br/>
-                 <p>The values in these tables were taken from, Markwardt et al. (2011)<sup>1</sup> and Jon&aacute;&scaron; et al.(2014)<sup>2</sup>.</p>
+                 <p>The values in these tables were taken from, Markwardt <i>et al.</i> (2011)<sup>1</sup> and Jon&aacute;&scaron; <i>et al.</i> (2014)<sup>2</sup>.</p>
                  <ul>
                      <li>Dilute samples equally if concentration is too high for the fluorescence reader of the machine.</li>
                      </ul>
                      <h3>Part 2</h3>
-                     <ul>
+                     <ol>
-                     <li>Add 90 uL of 1 mg/mL protein to 90 uL PBS for each protein in triplicate.</li>
+                     <li>Add 90 &micro;L of 1 mg/mL protein to 90 &micro;L PBS for each protein in triplicate.</li>
-                     <li>Add 90 uL of Cerulean to 90uL Venus in triplicate.</li>
+                     <li>Add 90 &micro;L of Cerulean to 90 &micro;L Venus in triplicate.</li>
-                     <li>Blank with 180 uL PBS.</li>
+                     <li>Blank with 180 &micro;L PBS.</li>
                      <li>Excite all wells at the ideal excitation value determined for Cerulean and scan wavelengths longer than this.</li>
                      <li>Please see <a target=_blank href=https://static.igem.org/mediawiki/2018/3/35/T--UNSW_Australia--FRET2018.zip>supplementary data</a> for specific parameters used.</li>
-                 </ul>
+                 </ol>
                  <br/>
      </div>
@@ Line 706: / Line 707: @@
                  <h3>Cellular Protocol</h3>
                      <ol>
-                         <li>Pick a single colony of untransformed T7 and transformed T7 and inoculate a 2-5ml overnight culture in LB. </li>
+                         <li>Pick a single colony of untransformed T7 and transformed T7 and inoculate a 2-5ml overnight culture in LB.</li>
-                         <li>Add antibiotics to the transformed culture. </li>
+                         <li>Add antibiotics to the transformed culture.</li>
-                         <li>The next day, add 50ml of LB + 2.5mg/mL tryptophan to a 250ml shake flask labelled “Control”. </li>
+                         <li>The next day, add 50 mL of LB + 2.5 mg/mL tryptophan to a 250 mL shake flask labelled “Control”. </li>
-                         <li>Add 50ml of LB + 2.5mg/mL tryptophan + antibiotic to another baffled shake flask labelled “IAA”. </li>
+                         <li>Add 50 mL of LB + 2.5 mg/mL tryptophan + antibiotic to another baffled shake flask labelled “IAA”. </li>
-                         <li>Add 1ml of the respective overnight cultures to each flask. Incubate with shaking (200 rpm) at 37&deg;C. Sample at 1h, 4h, 7h and 20-24h. </li>
+                         <li>Add 1 mL of the respective overnight cultures to each flask. Incubate with shaking (200 rpm) at 37&deg;C. Sample at 1h, 4h, 7h and 20-24h. </li>
                          <li>For each sample: a. Measure the OD at 600 nm.</li>
-                         <li>Spin down 1ml. Retain pellet and freeze. Retain supernatant and store at 4 C. Label these 4.</li>
+                         <li>Spin down 1 mL. Retain pellet and freeze. Retain supernatant and store at 4&deg;C. Label these 4.</li>
-                         <li>The next day:  Add Salkowski reagent to supernatants at the ratio of 1ml per 0.5ml of supernatant. Incubate in the dark for 30 minutes</li>
+                         <li>The next day:  Add Salkowski reagent to supernatants at the ratio of 1 mL per 0.5 mL of supernatant. Incubate in the dark for 30 minutes.</li>
-                         <li>Add 1ml of Salkowski reagent to each cell pellet. </li>
+                         <li>Add 1 mL of Salkowski reagent to each cell pellet.</li>
-                         <li>Vortex to mix. </li>
+                         <li>Vortex to mix.</li>
-                         <li>Incubate in the dark for 30 minutes. </li>
+                         <li>Incubate in the dark for 30 minutes.</li>
                          <li>Centrifuge at full speed for 5 minutes.</li>
-                         <li>Measure the absorbance at 530 nm Measure appropriate controls as well </li>
+                         <li>Measure the absorbance at 530 nm Measure appropriate controls as well.</li>
-                         <li> Normalise readings at each time point against OD600.</li>
+                         <li>Normalise readings at each time point against OD<sub>600</sub>.</li>
                      </ol>
                      <p>The assay is performed by adding the Salkowski reagent to a prepared sample:</p>
                          <ol>
-                             <li>To prepare the Salkowski assay reagent, slowly add 95% H2SO4 to MilliQ water in a ratio of 3:2, allowing time for heat dissipation between additions,</li>
+                             <li>To prepare the Salkowski assay reagent, slowly add 95 % H<sub>2</sub>SO<sub>4</sub> to Milli-Q water in a ratio of 3:2, allowing time for heat dissipation between additions.</li>
-                             <li>Dissolve 4.5g/L of anhydrous FeCl3 into the solution. Store in the dark, wrapped in foil.</li>
+                             <li>Dissolve 4.5 g/L of anhydrous FeCl<sub>3</sub> into the solution. Store in the dark, wrapped in foil.</li>
-                             <li>Prepare the standard by using a 3mM stock, then diluted 1 in 10 for usage – with 300uM to 0uM concentrations. </li>
+                             <li>Prepare the standard by using a 3 mM stock, then diluted 1 in 10 for usage – with 300 &micro;M to 0 &micro;M concentrations. </li>
                              <li>Prepare a standard curve.</li>
                              <li>Samples are run by adding the sample (or standard) to the Salkowski reagent in a ratio of 1:2 in a time sensitive manner within a 96-well microtitre plate. </li>
-                             <li>Run the plate on a plate reader for absorbance at 530nm, after being left for 30 minutes in the dark.</li>
+                             <li>Run the plate on a plate reader for absorbance at 530 nm, after being left for 30 minutes in the dark.</li>
                          </ol>
          </div>
@@ Line 734: / Line 735: @@
              <h2>High Performance Liquid Chromotography (HPLC)</h2>
                  <ol>
-                     <li>Acidify supernatant with 5M HCl to pH 2.5</li>
+                     <li>Acidify supernatant with 5 M HCl to pH 2.5.</li>
-                     <li>Extract supernatant with an equal volume of ethyl acetate</li>
+                     <li>Extract supernatant with an equal volume of ethyl acetate.</li>
-<li>Evaporate off the solvent by drying it through a vacuum (or leave it to evaporate).</li>
+                    <li>Evaporate off the solvent by either drying it through a vacuum or dissolving in methanol & acetic acid at pH 4.5 (25 % methanol, 1 % acetic acid, made up to 1 mL with H<sub>2</sub>O).</li>
-<li>Dissolve in methanol & acetic acid (pH 4.5)
+                    <li>250 &micro;L methanol, 10 &micro;L acetic acid, 740 &micro;L H<sub>2</sub>O.</li>
-<ol>
+                    <li>Run system with solution 72 % of 1 % acetic acid and 28 % of 100 % methanol for 0.8 mL/minute with injection of 30.</li>
-<li>1mL of a solution of 25% methanol, 1% acetic acid, rest H2O.</li>
-<li>250&micro;L methanol, 10&micro;L acetic acid, 740&micro;L H2O</li>
-</ol></li>
-<li>Run system with solution:
-	<ol>
-<li>72% of 1% acetic acid & 28% of 100% methanol.</li>
-<li>0.8mL/minute with injection of 30&micro;L.</li>
                  </ol>
          </div>
@@ Line 759: / Line 753: @@
          <h2>Seed Surface Sterilisation</h2>
              <ol>
-                 <li>Wild-type <i>Arabidopsis thaliana</i> seeds were surface sterilised by mixing in a solution of 2.5% sodium hypochlorite with a drop of Tween for 10 minutes.</li>
+                 <li>Wild-type <i>Arabidopsis thaliana</i> seeds were surface sterilised by mixing in a solution of 2.5 % sodium hypochlorite with a drop of Tween for 10 minutes.</li>
                  <li>Seeds were subsequently washed eight times in sterile water.</li>
              </ol>
-         <h2>Indole-3-aecetic Acid Filter Sterilisation</h2>
+         <h2>Indole-3-acetic Acid Filter Sterilisation</h2>
              <ol>
                  <li>10 mM stock indole-3-acetic acid (IAA) was prepared by dissolving indole-3-acetic acid sodium salt purchased from Sigma Aldrich in ethanol.</li>
-                 <li>In a laminar flow hood, the solution was filter sterilised through a 0.22μm Millex&reg; syringe driven filter and stored at 4&deg;C in a foil covered container to protect from light.</li>
+                 <li>In a laminar flow hood, the solution was filter sterilised through a 0.22 &micro;m Millex&reg; syringe driven filter and stored at 4&deg;C in a foil covered container to protect from light.</li>
              </ol>
          <h2>MS Agar Plate Preparation</h2>
@@ Line 771: / Line 765: @@
                  <li>Murashige and Skoog (MS) media was prepared using M5519 MS basal powdered medium purchased from Sigma Aldrich.</li>
                  <li>In four separate Schott bottles, 250 mL of MS media was prepared using 1.1 g of MS basal salts (per manufacturer instructions) and Milli-Q purified water.</li>
-                 <li>Each mixture was supplemented with sucrose (1% w/v) and agar powder (0.8% w/v) and adjusted to pH 5.7 using KOH.</li>
+                 <li>Each mixture was supplemented with sucrose (1 % w/v) and agar powder (0.8 % w/v) and adjusted to pH 5.7 using KOH.</li>
                  <li>Mixtures were autoclaved and allowed to cool to approximately 50&deg;C (or until able to be held comfortably).</li>
-                 <li>Cooled bottles were transferred to a dark laminar flow hood where indole-3-aecetic acid solution was added to prepare four separate MS agar mixtures at 0 μM, 1 μM, 10 μM and 100 μM concentrations IAA respectively.</li>
+                 <li>Cooled bottles were transferred to a dark laminar flow hood where indole-3-aecetic acid solution was added to prepare four separate MS agar mixtures at 0 &micro;M, 1 &micro;M, 10 &micro;M and 100 &micro;M concentrations IAA respectively.</li>
-                 <li>In a dark laminar flow hood, 50mL of media was pipetted into a square plate using a serological pipette. Four 50mL plates were prepared for each concentration of IAA.</li>
+                 <li>In a dark laminar flow hood, 50 mL of media was pipetted into a square plate using a serological pipette. Four 50 mL plates were prepared for each concentration of IAA.</li>
                  <li>Plates were labelled, sealed with parafilm and stored wrapped in foil at 4&deg;C until further use.</li>
              </ol>
@@ Line 780: / Line 774: @@
              <ol>
                  <li>MS media was prepared using M5519 MS basal powdered medium purchased from Sigma Aldrich.</li>
-                 <li>In an autoclavable Schott bottle, 500 mL 1/2 MS media was prepared using 1.1g of MS basal salts and 500 ml of Milli-Q purified water (4.4 g salts equivalent to 1L MS media per manufacturer’s instructions).</li>
+                 <li>In an autoclavable Schott bottle, 500 mL 1/2 MS media was prepared using 1.1 g of MS basal salts and 500 ml of Milli-Q purified water (4.4 g salts equivalent to 1 L MS media per manufacturer’s instructions).</li>
-                 <li>Media was supplemented with sucrose (1% w/v) and adjusted to pH 5.7 using KOH.</li>
+                 <li>Media was supplemented with sucrose (1 % w/v) and adjusted to pH 5.7 using KOH.</li>
                  <li>Media was autoclaved and stored at room temperature until further use.</li>
              </ol>
          <h2><i>Arabidopsis thaliana</i> Growth on Agar Plates</h2>
              <ol>
-                 <li>Surface sterilised seeds were added to a flask containing an autoclaved solution of Milli-Q water with 1% w/v sucrose in a laminar flow hood.</li>
+                 <li>Surface sterilised seeds were added to a flask containing an autoclaved solution of Milli-Q water with 1 % w/v sucrose in a laminar flow hood.</li>
                  <li>This flask was covered with foil and seeds were stratified at 4&deg;C for two days.</li>
                  <li>Stratified seeds were then allowed to germinate under light at room temperature for 2 days.</li>
@@ Line 795: / Line 789: @@
          <h2><i>Arabidopsis thaliana</i> Liquid Culture</h2>
              <ol>
-                 <li>In a laminar flow hood, 25 mL of 1/2 MS media with 1% sucrose was aliquoted into twelve 50 mL foil wrapped falcon tubes.</li>
+                 <li>In a laminar flow hood, 25 mL of 1/2 MS media with 1 % sucrose was aliquoted into twelve 50 mL foil wrapped falcon tubes.</li>
-                 <li>Filter sterilised IAA was added to prepare duplicate tubes at 0 nM, 1nM, 10 nM, 100 nM, 1 μM and 10 μM concentrations of IAA.</li>
+                 <li>Filter sterilised IAA was added to prepare duplicate tubes at 0 nM, 1nM, 10 nM, 100 nM, 1 &micro;M and 10 &micro;M concentrations of IAA.</li>
-                 <li>100 μL of surface sterilised <i>A. thaliana</i> seeds were added to each tube.</li>
+                 <li>100 &micro;L of surface sterilised <i>A. thaliana</i> seeds were added to each tube.</li>
                  <li>Liquid cultures were stratified at 4°C for two days, then grown under light at room temperature for 9 days.</li>
                  <li>Three seedlings from each tube were selected at random and measured for primary root length and shoot length.</li>
@@ Line 813: / Line 807: @@
          <li>Jon&aacute;&scaron;, A. et al. In vitro and in vivo biolasing of fluorescent proteins suspended in liquid microdroplet cavities. <i>Lab Chip</i> <b>14</b> 3093-3100 (2014).</li>
          <li>Tang, YW. and Bonner, J. The enzymatic inactivation of indoleacetic acid; some characteristics of the enzyme contained in pea seedlings. <i>Arch. Biochem.</i> <b>13</b> 11–25 (1947).</li>
-         <li>Glickmann, E. & Dessaux, Y. A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Applied and Environmental Microbiology <b>61</b>, 793–796 (1995).</li>
+         <li>Glickmann, E. and Dessaux, Y. A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Applied and Environmental Microbiology <b>61</b> 793–796 (1995).</li>
          <li>Xu, W. et al. An improved agar-plate method for studying root growth and response of <i>Arabidopsis thaliana</i>. <i>Sci Rep</i> <b>3</b> 1273 (2013).</li>
          </ol>

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

Restriction Enzyme	1 µL of each enzyme
DNA	1 µg
10X Cutsmart	5 µL (1X)
Total Reaction Volume	50 µL