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).

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-		<img height="150px" width="150px" class="top-icon" src="https://static.igem.org/mediawiki/2018/8/8d/T--UNSW_Australia--Icon-lab.png">
+            <img height="150px" width="150px" class="top-icon" src="https://static.igem.org/mediawiki/2018/8/8d/T--UNSW_Australia--Icon-lab.png">
-	</div>
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-	</div>
-	<h1 class="shadow-text main-heading">Experiments</h1>
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-	<div class="header-line">
+        <div class="bottom-icon-div">
-		<div id="lab-shadow-line" class="header-line-shadow"></div>
+            <img height="150px" width="150px" class="bottom-icon" src="https://static.igem.org/mediawiki/2018/f/f3/T--UNSW_Australia--Icon-lab-bw.png">
-	</div>
+        </div>
+        <h1 class="shadow-text main-heading">Experiments</h1>
+    </div>
+    <div class="header-line">
+        <div id="lab-shadow-line" class="header-line-shadow"></div>
+    </div>
 </div>
-<div id=experiments-content>
+<div id=experiments-content class="to-load">
-<div id=cloning>
+<br/>
-<h1>Cloning</h1>
+<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/>
+<div onclick="Cloning()" class="experiment-group">
+    <h2 class="shadow-text">DNA Cloning</h2>
+</div>
+    <div class="cloning subheading">
+<div id=cloning class="toggle-content">
 <div id=linearization class=protocol cloning>
-<h2>Linearisation pETDuet1 and pRSFDuet1 plasmid backbones using PCR</h2>
+<h2>Linearisation pETDuet1 and pRSFDuet1 Plasmid Backbones Using PCR</h2>
+<ol>
-<ul>
+<li>Forward and reverse primers created for the plasmids.</li>
-<li>Forward and reverse primers created for the plasmids</li>
+<li>2 &micro;L of plasmid + 198 &micro;L of water.</li>
-<li>2 uL of plasmid + 198 uL of water</li>
+<li>PCR amplification, according to the table below:</li>
-</ul>
+</ol>
-<table>
+<table class="lab-table">
 <thead>
 <tr>
 <th>Component</th>
 </td>
-<th>50 &micro;l Reaction</th>
+<th>50 &micro;L Reaction</th>
 </td>
 <th>Final Concentration</th>
@@ Line 49: / Line 55: @@
 </td>
 <td
-<p>25 &micro;l</p>
+<p>25 &micro;L</p>
 </td>
 <td
@@ Line 60: / Line 66: @@
 </td>
 <td>
-<p>2.5 &micro;l</p>
+<p>2.5 &micro;L</p>
 </td>
 <td>
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 </td>
 <td>
-<p>2.5 &micro;l</p>
+<p>2.5 &micro;L</p>
 </td>
 <td>
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 </td>
 <td>
-<p>2 uL DILUTED</p>
+<p>2 &micro;L diluted</p>
 </td>
 <td>
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 </td>
 <td>
-<p>18 uL</p>
+<p>18 &micro;L</p>
 </td>
 <td>&nbsp;</td>
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 <thead>
 <tr>
-<th>STEP</th>
+<th>Step</th>
-<th>TEMP</th>
+<th>Temperature</th>
-<th>TIME</th>
+<th>Time</th>
 </tr>
 </thead>
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 <tr>
 <td>
-<p>62</p>
+<p>62&deg;C</p>
 </td>
 <td>
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 </td>
 <td>
-<p>2 min</p>
+<p>2 minutes</p>
 </td>
 </tr>
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 </table>
 </div>
+<br/>
-</div>
 <div id=dpn1 class=protocol cloning>
-<h2>Plasmid digest (Dpn1 digest)</h2>
+<h2>Plasmid Digest (Dpn1 Digest)</h2>
-<ul>
+<ol>
 <li>Set-up the reaction mixture:
-<table>
+<table class="lab-table">
 <tbody>
 <tr>
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 </td>
 <td>
-<p>1&micro;l</p>
+<p>1 &micro;L</p>
 </td>
 </tr>
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 <td>
-<p>5 &micro;l (1X)</p>
+<p>5 &micro;L (1X)</p>
 </td>
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 </td>
 <td>
-<p>50 &micro;l</p>
+<p>50 &micro;L</p>
 </td>
 </tr>
 </tbody>
 </table></li>
-<li>Incubate for 1 hr at 37C</li>
+<li>Incubate for 1 hr at 37&deg;C.</li>
-<li>Heat inactivate at 80C for 20 minutes</li>
+<li>Heat inactivate at 80&deg;C for 20 minutes.</li>
 </ol>
 </div>
+<br/>
 <div id=gel class=cloning protocol>
-<h2>Agarose gel electrophoresis</h2>
+<h2>Agarose Gel Electrophoresis</h2>
-<ul>
+<ol>
-	<li>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.</li>
+<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 (do not overboil the solution). Stop and swirl the flask every 20 seconds and until the solution is as clear as water.</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>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.</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 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.</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 box, fill the gel box with 1 x TAE buffer until the gel is covered then remove the well comb.</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 uL of the digest sample with 3 uL of H2O and 1 uL of 6x loading dye.</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>
-	<li>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).</li>
+<li>Load DNA ladder into the first well of the gel, followed by your DNA samples.</li>
-	<li>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.</li>
+<li>Connect the gel tank to a power pack and run the gel at 100 V for 1 hr.</li>
-	<li>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.</li>
+<li>Carefully take the gel tray to a Gel Doc for imaging.</li>
-</ul>
+</ol>
 </div>
+<br/>
 <div id=gibson class=cloning protocol>
 <h2>Gibson Assembly</h2>
 <h3>Materials:</h3>
-<ul>
+<p>5X Isothermal Reaction Mix (6 mL total, Store at -20&deg;C):</p>
-<li>5X Isothermal Reaction Mix (6 mL total)</li>
+<table class="lab-table">
-	<li>3 mL 1 M Tris-Hcl (pH 7.5)</li>
+<tbody>
-	<li>300 μL 1 M MgCl2</li>
+<tr>
-	<li>60 μL 100 mM dGTP</li>
+<td>1 M Tris-HCl (pH 7.5)</td>
-	<li>60 μL 100 mM dATP</li>
+<td>3 mL</td>
-	<li>60 μL 100 mM dTTP</li>
+</tr>
-	<li>60 μL 100 mM dCTP</li>
+<tr>
-	<li>300 μL 1 M DTT</li>
+<td>1 M MgCl<sub>2</sub></td>
-	<li>1.5 g PEG-8000</li>
+<td>300 &micro;L</td>
-	<li>300 μL 100 mM NAD</li>
+</tr>
-	<li>360 µL water</li>
+<tr>
-	<li>Store at -20°C.</li>
+<td>100 mM dGTP</td>
-</ul></li>
+<td>60 &micro;L</td>
-<li>Assembly Master Mix (1.2 mL total)
+</tr>
-<ul>
+<tr>
-	<li>320 μL 5X Isothermal Master Mix</li>
+<td>100 mM dATP</td>
-	<li>0.64 μL 10 U/μL T5 exonuclease</li>
+<td>60 &micro;L</td>
-	<li>20 μL 2 U/μL Phusion DNA Pol</li>
+</tr>
-	<li>0.16 μL 40 U/μL T4 DNA Ligase</li>
+<tr>
-	<li>860 μL water</li>
+<td>100 mM dTTP</td>
-	<li>Store in 15 µL aliquots at -20°C.</li>
+<td>60 &micro;L</td>
-</ul></li>
+</tr>
-<ul>
+<tr>
-	<li>PCR or digest your fragment of choice and gel purify</li>
+<td>100 mM dCTP</td>
-	<li>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.</li>
+<td>60 &micro;L</td>
-	<li>Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.</li>
+</tr>
-	<li>Add 5 μl of DNA to be assembled to the master mixture. </li>
+<tr>
-	<li>The DNA fragments should be in equimolar amounts.</li>
+<td>1 M DTT</td>
-	<li>Small fragments (<1 kb) must be added in a five times excess</li>
+<td>300 &micro;L</td>
-	<li>You can calculate the quantity of each fragment using their molecular weights.</li>
+</tr>
-	<li>Alternatively, you can use the length of each fragment as a proxy for the molecular weight (assuming similar GC content in all fragments).</li>
+<tr>
-	<li>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).</li>
+<td>PEG-8000</td>
-	<li>Incubate at 50 °C for 15 to 60 min (60 min is optimal).</li>
+<td>1.5 g</td>
-</ul>
+</tr>
+<tr>
+<td>100 mM NAD</td>
+<td>300 &micro;L</td>
+</tr>
+<tr>
+<td>H<sub>2</sub>O</td>
+<td>360 &micro;L</td>
+</tr>
+</tbody>
+</table>
+<br/>
+<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 &micro;L</td>
+</tr>
+<tr>
+<td>510 U/&micro;L T5 exonuclease</td>
+<td>0.64 &micro;L</td>
+</tr>
+<tr>
+<td>2 U/&micro;L Phusion DNA Polymerase</td>
+<td>20 &micro;L</td>
+</tr>
+<tr>
+<td>40 U/&micro;L T4 DNA Ligase</td>
+<td>0.16 &micro;L</td>
+</tr>
+<tr>
+<td>H<sub>2</sub>O</td>
+<td>860 &micro;L</td>
+</tr>
+</tbody>
+</table>
+<br/>
+<h3>Method:</h3>
+<ol>
+<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 &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 &micro;L.</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 minutes (60 minutes is optimal).</li>
+</ol>
 </div>
+<br/>
 <div id=heat-shock class=protocol cloning>
-<h2>Heat shock transformation</h2>
+<h2>Heat Shock Transformation</h2>
-<ul>
+<ol>
-	<li>Incubate 50ng of plasmid construct  with 25 µL of chemically competent <i>E. coli</i> T7</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>Express cells or <i>E. coli</i> 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>Heat shock the cells for 45 seconds at 42C and placed back onto ice for 2 minutes. </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>Allow cells to grow for 45 minutes in 200 µL of SOC outgrowth media (NEB) at 37°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>
-	<li>Spread plate onto Luria broth (LB) agar plates containing 100 µg/mL of ampicillin and grown at 37°C overnight.</li>
+</ol>
-</ul>
 </div>
+<br/>
 <div id=colonyPCR class=protocol cloning>
 <h2>Colony PCR</h2>
-<li>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.</li>
+<ol>
-<li>Create the colony PCR master mix. Determine volumes for the cPCR master mix by multiplying (the number of reactions + 2) by each volume below:
+<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>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.</li>
+</ul>
-<li>Add 1 uL of each bacterial dilution and 24 uL of cPCR master mix to a PCR tube.</li>
+</li>
-<li>Run PCR with the following instructions (lid at 105 C and volume = 20 uL):
+<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 &micro;L):
 <ul>
-<li>95 C 5:00 mins</li>
+<li>Step 1: 95&deg;C for 5 minutes</li>
-<li>95 C 0:30 mins</li>
+<li>Step 2: 95&deg;C for 30 seconds</li>
-<li>55 C 0:30 mins</li>
+<li>Step 3: 55&deg;C for 30 seconds</li>
-<li>68 C 2:00 mins</li>
+<li>Step 4: 68&deg;C for 2 minutess</li>
-<li>68 C 5:00 mins</li>
+<li>Repeat 25-35 cycles of steps 2-4</li>
-<li>4 C 0:00 mins (hold)</li>
+<li>Step 5: 68&deg;C 5 minutes</li>
+<li>Step 6: Hold at 4&deg;C</li>
 </ul></li>
-<li>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.</li>
+<li>Run PCR products on a 1 % agarose gel and image for analysis.</li>
+</ol>
 </div>
+<br/>
 <div id=sequencing class=protocol cloning>
 <h2>Sequencing</h2>
-<ul>
+<ol>
-	<li>Transfer 10 uL of purified plasmid sample (50-100ng/uL) to an Eppendorf tube. Add 5 uL of one primer.</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. http://sequencing.ramaciotti.unsw.edu.au/index.html</li>
+<li>Request sequencing at the UNSW Centre for Genomics via the on-line portal.</li>
-	<li>Label Eppendorf tubes with the order number, and 1, 2, 3, etc.</li>
+<li>Label PCR tubes carefully with numbers as per the on-line order.</li>
-	<li>Take Eppendorf tubes to the new Biosciences building Lvl 2 (at UNSW, Sydney, Australia), and store the samples in the fridge provided.</li>
+<li>Take PCR tubes to the Ramaciotti Centre for Genomics Lvl 2 (at UNSW, Sydney, Australia), and store the samples in the fridge provided.</li>
-	<li>Sanger sequencing is carried out following <a target=_blank href=http://www.ramaciotti.unsw.edu.au/wp-content/uploads/2016/09/RAMAC_LIMS_User_Guide-2016.pdf>the provided protocol</a>.</li>
+<li>Sanger sequencing is carried out by the Centre staff following <a target=_blank href=http://www.ramaciotti.unsw.edu.au/wp-content/uploads/2016/09/RAMAC_LIMS_User_Guide-2016.pdf>the provided protocol</a>.</li>
-</ul>
+</ol>
 </div>
+<br/>
 <div id=restriction class=cloning protocol>
-<h2>Restriction cloning</h2>
+<h2>Restriction Cloning</h2>
-<ul>
+<ol>
-<li>Set-up the reaction
+<li>Set-up the reaction mixture:
-<table>
+<table class="lab-table">
 <tbody>
 <tr>
@@ Line 321: / Line 379: @@
 </td>
 <td>
-<p>1&micro;l of each enzyme</p>
+<p>1 &micro;L of each enzyme</p>
 </td>
 </tr>
@@ Line 337: / Line 395: @@
 </td>
 <td>
-<p>5 &micro;l (1X)</p>
+<p>5 &micro;L (1X)</p>
 </td>
 </tr>
@@ Line 345: / Line 403: @@
 </td>
 <td>
-<p>50 &micro;l</p>
+<p>50 &micro;L</p>
 </td>
 </tr>
@@ Line 351: / Line 409: @@
 </table>
 </li>
-<li>Incubate for 1 hr at 37C</li>
+<li>Incubate for 1 hr at 37&deg;C.</li>
-<li>Heat inactivate at 80C for 20 minutes</li>
+<li>Heat inactivate at 80&deg;C for 20 minutes.</li>
-</ul>
+</ol>
 <h3>Ligation</h3>
+<ol>
 <li>Set up the following reaction in a microcentrifuge tube on ice:
+<table class="lab-table">
-<table>
 <tbody>
 <tr>
 <th>Component</th>
-<th>20 μl Reaction</th>
+<th>20 &micro;L Reaction</th>
-</td>
 </tr>
 <tr>
@@ Line 369: / Line 425: @@
 <p>T4 DNA Ligase Buffer (10X)*</p>
 </td>
-<p>2 μl</p>
+<td>
+<p>2 &micro;L</p>
+</td>
 </tr>
 <tr>
@@ Line 392: / Line 450: @@
 </td>
 <td>
-<p>to 20 μl</p>
+<p>to 20 &micro;L</p>
 </td>
 </tr>
@@ Line 400: / Line 458: @@
 </td>
 <td>
-<p>1 μl</p>
+<p>1 &micro;L</p>
 </td>
 </tr>
 </tbody>
 </table></li>
 <li>Gently mix the reaction by pipetting up and down and microfuge briefly.</li>
-<li>For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.</li>
+<li>For cohesive (sticky) ends, incubate at 16&deg;C overnight or room temperature for 10 minutes.</li>
-<li>Heat inactivate at 65°C 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>
+<br/>
 <div id=miniprep class=protocol cloning>
 <h2>Miniprep</h2>
-<ul>
+<p>Protocols were followed from the Qiagen QIAprep Spin Miniprep Kit Cat No./ID: 27104. No changes were made.</p><br/>
-<li>Protocols were followed from the Qiagen QIAprep Spin Miniprep Kit. No changes were made.</li>
-</ul>
 </div>
 </div>
+    </div>
-<div id=protein>
+<div onclick="Protein()" class="experiment-group">
-<h1>Protein Expression and Purification</h1>
+    <h2 class="shadow-text">Protein Production</h2>
-<div id=starter-culture class=protocol protein>
-<h2>Starter culture</h2>
-<ul>
-<li>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.</li>
-</ul>
 </div>
+    <div class="protein subheading">
+<div id=protein class="toggle-content">
+        <div id=starter-culture class=protocol protein>
+            <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&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&deg;C are inoculated with the starter culture.</li>
+                    <li>The cells are grown at 37&deg;C and 200 rpm and OD<sub>600</sub> is periodically measured. </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&deg;C, 200 rpm.</li>
+                </ol>
+        </div>
+        <div id=cell-collection class=protocol protein>
+            <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 (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>
+                <ol>
+                    <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>
+                </ol>
+        </div>
+        <div id=IMAC class=protocol protein>
+            <h2>IMAC</h2>
+                <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 (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 (20 mM NaH<sub>2</sub>PO<sub>4</sub>, 500 mM NaCl, 500 mM Imidazole).</li>
+                    <li>Analyse fractions with SDS-PAGE.</li>
+                </ol>
+        </div>
+        <div id=buffer-exchange class=protocol protein>
+            <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>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 0.01 is reached. That is, until there is 1% of the old buffer left in the solution.</li>
+                </ol>
+                <h3>Dialysis</h3>
+                <ol>
+                    <li>Add 1 mL of protein and 1 mL of PBS buffer (pH 8) to a 15 mL Falcon tube.</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>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>
+                </ol>
+        </div>
+        <div id=western class=protocol protein>
+            <h2>Western Blot</h2>
+                <h3>Materials</h3>
+                    <ul>
+                        <li>NuPAGE Bis-Tris gel</li>
+                        <li>NuPAGE MES running buffer</li>
+                        <li>Mini iBlotTM stack</li>
+                        <li>TBS-T: </li>
+                        <li>1X TBS with 0.1 % Tween20</li>
+                        <li>Blocking Solution: </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>Chemiluminescent HRP substrate</li>
+                    </ul>
+                <h3>Sample Preparation</h3>
+                    <ol>
+                        <li>Add reducing buffer to the bacterial lysates.</li>
+                        <li>Heat at 95&deg;C for 5 minutes.</li>
+                    </ol>
+                <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>Place the gel inside the tank, and fill with NuPAGE MES running buffer.</li>
+                        <li>Load 5 &micro;L of the protein standards ladder into the first well.</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 160 V for 40 minutes.</li>
+                    </ol>
+                <h3>Protein Transfer</h3>
+                    <ol>
+                        <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>Run at 20 V for 7 minutes.</li>
+                    </ol>
+                <h3>Blocking</h3>
+                    <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&deg;C overnight, or at room temperature for 2 hours.</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>Prepare HRP substrate according to manufacturer's instructions and add to the membrane.</li>
+                        <li>Image.</li>
+                    </ol>
+        </div>
+    </div>
+    </div>
-<div id=grow-up class=protocol protein>
+<div onclick="Assembly()" class="experiment-group">
-<h2>Large-scale grow-up</h2>
+    <h2 class="shadow-text">Assembly</h2>
-<ul>
-<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>The cells are grown at 37°C and 200 rpm and OD600 is periodically measured. </li>
-<li>Once OD600 reaches above 0.6, add IPTG of 1 mM concentration to induce the expression the proteins.</li>
-<li>After induction, grow the cells overnight at 24°C, 200 rpm.</li>
-</ul>
 </div>
+    <div class="assembly subheading">
-<div id=cell-collection class=protocol protein>
+<div id=assembly class="toggle-content">
-<h2>Collection of cells by centrifugation</h2>
+        <div id=catcher-tag class=protocol assembly>
+            <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>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 &micro;L sample was obtained.</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>
+        </div>
+        <div id=SEC class=protocol assembly>
+            <h2>Size Exclusion Chromotography (SEC)</h3>
+            <p>We would like to thank Hélène Lebhar of the UNSW Recombinant Proteins facility for conducting SEC experiments for us.</p>
+        </div>
+        <div id=TEM class=protocol assembly>
+            <h2>TEM</h2>
+                 <h3>Materials</h3>
 <ul>
-<li>Centrifuge the bacterial culture at 4600 x g for 20 minutes.</li>
+<li>Protein sample (ideally in water or low salt buffer)</li>
-<li>Collect cell pellet and resuspended in binding buffer (20mm NaH2PO4, 500mM NaCl, 10mM Imidazole).</li>
+<li>Milli-Q water</li>
+<li>2 % uranyl acetate</li>
+<li>Parafilm</li>
+<li>Self-closing forceps</li>
 </ul>
-</div>
+<h3>Staining</h3>
+<ol>
+<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 7 &micro;L of sample on a grid.</li>
+<li>Let the grid stain for 10 minutes.</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>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 5-7 minutes.</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>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>
+</ol>
+        </div>
+    </div>
+    </div>
-<div id=lysis class=protocol protein>
+<div onclick="Fret()" class="experiment-group">
-<h2>Cell lysis by sonication</h2>
+    <h2 class="shadow-text">FRET</h2>
-<ul>
-<li>Lyse the cell pellet by sonication (Branson) for 10 minutes at 50% amplitude at 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>
-</ul>
 </div>
+    <div class="fret subheading">
+<div id=FRET class="toggle-content">
+            <h2>FRET Protocol for Negative Controls</h2>
+                <h3>Part 1</h3>
+                <ol>
+                    <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 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 <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 &micro;L PBS.</li>
+                </ol>
+                <br/>
+                    <table class="lab-table">
+                    <tr>
+                    <th></th>
+                    <th>Excitation</th>
+                    <th>Emission</th>
+                    </tr>
+                    <tr>
+                    <td>mCerulean3</td>
+                    <td>433 (400-465)</td>
+                    <td>475 (465-525)</td>
+                    </tr>
+                    <tr>
+                    <td>mVenus</td>
+                    <td>515 (500-525)</td>
+                    <td>528 (520-550)</td>
+                    </tr>
+                    </table>
+                <br/>
+                <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>
+                    <ol>
+                    <li>Add 90 &micro;L of 1 mg/mL protein to 90 &micro;L PBS for each protein in triplicate.</li>
+                    <li>Add 90 &micro;L of Cerulean to 90 &micro;L Venus in triplicate.</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>
+                </ol>
+                <br/>
+    </div>
+    </div>
-<div id=IMAC class=protocol protein>
+<div onclick="Assays()" class="experiment-group">
-<h2>IMAC</h2>
+    <h2 class="shadow-text">Enzyme Assays</h2>
-<p>(Immobilised metal ion affinity chromatography (IMAC) was performed to purify the expressed proteins.)</p>
-<ul>
-<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>Elute with 2 mL of elution buffer (same as binding, but with 500 mM imidazole).</li>
-<li>Collect fractions with SDS-PAGE.</li>
-</ul>
 </div>
+    <div class="assays subheading">
+    <div id=enzyme-assays class="toggle-content">
+        <div id=BCA class=protocol assays>
+            <h2>BCA Protein Assay</h2>
+                <p>Protocol followed as per Pierce BCA Protein Assay Kit 23225 to determine protein concentration in solution:</p>
+                <ul>
+                    <li>Sample to working reagent ratio =  1:8</li>
+                    <li>96-well plate reader: SPECTROstar Nano BMG LABTECH</li>
+                    <li>Software used: SPECTROstar Nano</li>
+                </ul>
+        </div>
+        <div id=salkowski class=protocol assays>
+            <h2>Salkowski Assay</h2>
+            <p>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 <a href=https://2011.igem.org/Team:Imperial_College_London/Project_Auxin_Design>2011 iGEM Imperial College London</a>, Tang and Bonner’s 1947 paper<sup>3</sup>, and Glickmann and Dessaux's 1995 paper<sup>4</sup>.</p>
+                <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>Add antibiotics to the transformed culture.</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 50 mL of LB + 2.5 mg/mL tryptophan + antibiotic to another baffled shake flask labelled “IAA”. </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 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 1 mL per 0.5 mL of supernatant. Incubate in the dark for 30 minutes.</li>
+                        <li>Add 1 mL of Salkowski reagent to each cell pellet.</li>
+                        <li>Vortex to mix.</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>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 % 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 4.5 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 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 530 nm, after being left for 30 minutes in the dark.</li>
+                        </ol>
+        </div>
+        <div id=HPLC class=protocol assays>
+            <h2>High Performance Liquid Chromotography (HPLC)</h2>
+                <ol>
+                    <li>Acidify supernatant with 5 M HCl to pH 2.5.</li>
+                    <li>Extract supernatant with an equal volume of ethyl acetate.</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>250 &micro;L methanol, 10 &micro;L acetic acid, 740 &micro;L H<sub>2</sub>O.</li>
+                    <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>
+                </ol>
+        </div>
+    </div>
+    </div>
-<div id=buffer-exchange class=protocol protein>
+<div onclick="Plants()" class="experiment-group">
-<h2>Buffer exchange</h2>
+    <h2 class="shadow-text">Plants</h2>
-<h3>Column</h3>
-<ul>
-	<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>Top up column 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, there is 1% of the old buffer left in the solution.</li>
-</ul>
-<h3>Dialysis</h3>
-<ul>
-	<li>Add 1mL of protein and 1mL of PBS buffer (PH 8) to a 15mL Falcon tube.</li>
-	<li>Add 2mL 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>Add 500mL of PBS buffer, pH 8, (this is the buffer we want to exchange into) into a 500ml 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>
-</ul>
 </div>
+    <div class="plants subheading">
-<div id=western class=block protocol>
+    <div id="plants-content" class="toggle-content">
-    <h2>Western Blot</h2>
+        <h2>Seed Surface Sterilisation</h2>
-        <h3>Materials</h3>
-            <ul>
-                <li>NuPAGE Bis-Tris gel</li>
-                <li>NuPAGE MES running buffer</li>
-                <li>Mini iBlotTM stack</li>
-                <li>TBS-T: </li>
-                <li>1x TBS with 0.1% Tween20</li>
-                <li>Blocking Solution: </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>Chemiluminescent HRP substrate</li>
-            </ul>
-        <h3>Sample Preparation</h3>
              <ol>
-                 <li>Add reducing buffer to the bacterial lysates</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>Heat at 95 degrees for 5 minutes</li>
+                 <li>Seeds were subsequently washed eight times in sterile water.</li>
              </ol>
-         <h3>SDA-Page Gel</h3>
+         <h2>Indole-3-acetic Acid Filter Sterilisation</h2>
              <ol>
-                 <li>Remove the NuPAGE gel from its packaging and peel off the plastic strip from its base</li>
+                 <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>Place the gel inside the tank, and fill with NuPAGE MES running buffer</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>
-                <li>Load 5uL of the protein standards ladder into the first well</li>
-                <li>Load up to 20uL of each lysate sample into the wells</li>
-                <li>Connect the gel tank to a power pack, and run at 160V for 40min</li>
              </ol>
-         <h3>Protein Transfer</h3>
+         <h2>MS Agar Plate Preparation</h2>
              <ol>
-                 <li>Remove and rinse the gel in water</li>
+                 <li>Murashige and Skoog (MS) media was prepared using M5519 MS basal powdered medium purchased from Sigma Aldrich.</li>
-                 <li>Inside an iBlotTM Transfer Device, assemble the mini stack with the gel inside</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>Run at 20V for 7min</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 &micro;M, 1 &micro;M, 10 &micro;M and 100 &micro;M concentrations IAA respectively.</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>
-         <h3>Blocking</h3>
+         <h2>Liquid Culture Media Preparation</h2>
-            <ol><li>Incubate the membrane for 1-2hrs 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>MS media was prepared using M5519 MS basal powdered medium purchased from Sigma Aldrich.</li>
-                 <li>Wash the membrane in TBS-T three times for 10mins per wash at room temperature, shaking</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 autoclaved and stored at room temperature until further use.</li>
              </ol>
-         <h3>Detection</h3>
+         <h2><i>Arabidopsis thaliana</i> Growth on Agar Plates</h2>
              <ol>
-                 <li>Remove membrane from the last wash and place in chemiluminescent image analyser</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>Prepare HRP substrate according to manufacturer's instructions and add to the membrane</li>
+                 <li>This flask was covered with foil and seeds were stratified at 4&deg;C for two days.</li>
-                 <li>Image</li>
+                <li>Stratified seeds were then allowed to germinate under light at room temperature for 2 days.</li>
+                <li>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.</li>
+                <li>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 illumination<sup>5</sup>. Plates were stored upright at an approximately 90&deg; angle. The plated seedlings were grown under light at room temperature for 20 days.</li>
+                 <li>Primary root length and lateral root numbers were measured at 4, 6, 11 and 20 days following the initial seedling transplant.</li>
              </ol>
-</div>
+        <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>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 &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>
+            </ol>
+    </div>
+    </div>
+    <div id=references>
+<br/>
+    <h2>References</h2>
+        <ol>
+        <li>Markwardt, M. et al. An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching. <i>PLOS ONE</i> <b>6</b> e17896 (2011).</li>
+        <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. 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>
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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