Difference between revisions of "Team:DTU-Denmark/Experiments"

Latest revision as of 07:24, 20 October 2019

Experiments

If you've ever participated in iGEM, then you know just how many hours we have spent in the lab. Even though we didn't quite install half a dozen beds in the break room like we wanted to, we still felt like sharing our experiences with all of you. Behold! Our protocols.

Protocols

Lab friendly downloads available for all protocols

This protocol is adapted from a protocol used by DTU Bioengineering.

Materials

Fungal plates (either streaked or 3-point)
Drigalski spatula
500 mL shake flasks
Counting chamber
Solutions (APB, ATP, PCT, Milli-Q, TM)
30 °C incubator with shaking
Sterile tea spoon
Mira cloth in funnel (sterile)
Glucanex
Magnet stirrer
Magnets
50 mL sterile falcon tubes
0.45 µm filters
50 mL syringe
Centrifuge for falcon tubes

Media

Aspergillus transformation buffer (ATB)
Aspergillus protoplastation buffer (APB)
500 mL shake flasks
YPD media

Procedure

Initiation

Streak spore suspension of host strain on YPD plates supplemented with uridine (for this particular fungus) and let grow for a week (there should be black spores!)
All solutions should be sterile.

Day 1 (inoculation)

Add 95 mL of YPD media supplemented with uridine to a shake flask and transfer 5 mL of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 mL. It is a good idea to make more than one shake flask at a time.
Incubate shake flasks at 30 °C, 150 RMP for 48 h.

Day 3 (mycelial harvest)

Place the sterile funnel with a mira cloth in to a sterile blue cap bottle and transfer the contents of the shake flasks to the mira cloth (content should be brown and thick).
Wash the mycelia using Aspergillus protoplastation buffer (APB) to remove residual glucose from the mycelia (this can inhibit protoplastation). You need to use quite a bit of APB. squeeze out remaining liquid using a sterile spoon. Then, transfer the mycelium to falcon tubes ( ≈2 g per tube => 1 shake flask ≈ 2 falcon tubes).

Protoplastation

Add glucanex to APB to get a final concentration of 40 mg glucanex per mL APB and dissolve glucanex via gentle magnetic stirring and no heat.
Sterile filter 20 mL of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 mL syringe (there is a bit of resistance in the filter but that's ok).
Shake/incubate enzyme-mycelium mix at 30 °C, 150 rpm for 2-3 h.
From now on, whenever you pipette anything with the cells in it, cut of the edge of the pipette tip and CAREFULLY(!) pipette the cells. If you don't do this, they break as they don't have a cell wall to keep them stable.
Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/mL). Approved protoplast solutions are then diluted by pouring APB up to 40 mL. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile Milli-Q water and carefully place 5 mL of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)
In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 mL and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.
Count protoplasts in microscope by diluting a small sample 1:100.
Resuspend protoplasts in 4 mL ATB to obtain concentrated solution.

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A quick overview of the two different transformation protocols

Materials

DNA
Protoplasts
Falcon tubes
PCT
TM molten agar
TM plates
ATB

Procedure

Regular protocol

Add at MOST 20 µL DNA ( or max 25% of protoplast volume) and 100 µL of protoplasts in a 50 mL falcon tube.
Incubate in the falcon tube at RT for at least 30 min.
Add 1mL of PCT.
Gently mix by gently swirling the tube in a circular motion – careful they are fragile. Do not vortex or pipette mix.
Incubate for 5 min at RT.
Add 3 mL of ATB.
Add 12 mL of molten (40-45 °C) TM agar.
Immediately pour mixture directly onto TM plates, and swirl to spread mixture evenly.
Let the plates settle for a few minutes and incubate at 37 °C for 4 days.

Quick protocol

Add at MOST 25 µL (1500-5000 ng) DNA ( or max 25% of protoplast volume) and 100 µL of protoplasts in a 2 mL Eppendorf tube. 1 µL (100 ng) pac6 as positive control.
Add 150 μL PCT with large-nozzle pipette tip.
Gently mix by swirling – careful the protoplasts are fragile.
Incubate 10-30 min at room temperature.
Add 250 μL ATB.
Distribute transformation mix on osmotic-stabilized selective media and let the agar absorb the mix before incubating

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Protocol for producing a glycerol stock of E. coli adapted by Jacob Mejlsted from Addgene's protocol.

Materials

Consumables

2 mL screw top tube or cryovial

Chemicals

50% glycerol
Cell culture

Procedure

Making the stock

After having cultivated a culture overnight, add 500 µL culture to 500 µL 50% glycerol in the selected tube
The stock can now be frozen at -80 °C
If the stock is repeatedly thawed and frozen, it will reduce the shelf life of the culture

Using the stock

To recover bacteria, take a innoculation loop, toothpick or pipete tip and scrape a small amount of bacteria off the top.
Transfer this to a plate or a tube with liquid media for growth.
Normally, media with antibiotics are used. Here Amp and Cam are among the most used.

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How to load and clean biolector plate.

Materials

Biolector
Minimal media
P1000 pipette and tips
P10 pipette and tips
Spore suspensions
Biolector plate
Lid for the biolector plate

Procedure

NB! Make sure to do all work in a clean LAF-bench!
Clean plate with ethanol and a cotton swab and leave to dry
Clean additionally using UV for >30 min.
Add 1500 µl minimal media (+supplements if necessary) to each well.
Add spores to the wells to achieve a final concentration of 10⁶ or 10⁷ spores per mL.
Add a lid for the plate. These are sticky and should be breathable, i.e. the white, kind of fabric-y lids
Run Biolector for 36 hours
After run, fill all wells with ethanol and clean using "soft" things such as a pipette tip to get to the corners and a cue tip to clean the sides. Repeat this process until the plate is clean.

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Protocol for preparation and execution of the 1 L bioreactor (Sartorius Stedim 1L) cultivations. Adapted from DTU course material by Helena Utzon.

Materials

1 L Sartorius Stedim 1L bioreactor
Sartorius Stedim BIOSTAT Q Plus control tower
Off-gas
Minimal media
Sigma 204 Antifoam
Sterile arginine solution
Acid (2 M Sulfuric acid)
Base (2 M Potassium hydroxide)
Spore suspensions

Consumables

1 mL syringes
Lab grease
White strips

Procedure

Assembly of the bioreactors

Mount the inoculation, sampling, and temperature port from the bottom of the lid and secure them with the bolt from the top of the lid.
Mount the impellers at the correct height.
Attach the sparger and baffles, and secure them with the bolt at the top of the bioreactor.
Mount the condenser, preferably as close to the control tower as possible.
The mouth of the condenser is loosely packed with a bit of glass wool, and the condenser is closed off.
The temperature and sampling pipe is mounted, and the sampling tubing is attached and sealed off with both a butterfly clamp and a regular clamp.
The inoculation port (with membrane) is assembled and mounted.
The 4-way connector is attached to the bioreactor, and the un-needed connections are closed off with a knotted piece of tubing.
Marprene tube is attached to the air inlet, and an air filter is attached to the tubing in the right direction.
Assemble the bioreactor, using a bit of lab grease on the rubber gaskets.
Connect the empty 250 mL Blue Cap bottles (acid/base) to the 4-way connector, with tubing appropriate for the control tower pumps.
Clamp tubing between the bioreactor and bottles, as close to the reactor as possible. Also, attach a clamp the tube leading to the air filter.
Secure all tubing with white strips.
Fill the reactor with 1 L of minimal medium (see media recipe).
Calibrate the pH-electrode, and pressure test the reactor.
Cover all open ends and filters with tin foil to protect them during autoclaving.
Check all clamps prior to autoclavation, and loosen the main screw to prevent overpressure.
Autoclave the bioreactor.
After autoclavation, close the loose fitting and mount the air supply. Set the flow rate on the DCU to 0.75 vvm, and remove all clamps except for on the sampling tubing.
Once the medium has cooled down, connect the condenser tubing, the temperature jacket tubing, pH-electrode, DO-electrode, and the stirrer.
Set the stirrer to 800 rpm and check the accuracy of the pH-electrode. Calibrate if needed.
Mount the tubing for the acid and base on their respective pumps, run the acid/base through the tubing and switch to “auto”.
Calculate the inoculum volume to a final concentration of 10⁶ spores/mL, and inoculate the bioreactors with the previously prepared spore suspensions (See Here).
Add 50 µL of Sigma 204 Antifoam to the reactor.
Due to species limitations, 0.7 g of arginine was added to the media of the positive control.
Set the timer to zero at the DCU, and start the batch fermentation at the desired process values.
Please find the ramps and process conditions for our bioreactor cultivations below.

Bioreactor conditions

Process conditions
Airflow inlet
Time (min)	ValueVVM (L/L/M)
0	0.1
300	0.16
420	0.4
600	1
pH
Time (min)	ValuepH
0	3
600	5
Stirrer speed
Time (min)	ValueRPM
0	100
300	200
420	300
600	500
720	800
Temperature
Time (min)	Value(°C)
Start-end	30

Sampling can then be performed as described in the sampling protocol

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By David Faurdal, adapted in part from NEB's one-taq protocol.

The purpose of this protocol is to confirm correct insertion of fragments after assemblies, such as 3A, Gibson, or Golden Gate. As the fragments run on the gel won't be used for cloning purposes, there is no reason to use high-fidelity polymerases on this, just use one-taq.

Materials

Transformants from whatever assembly method you fashion
Eppendorf tubes
Sterile toothpicks/inoculation lops/pipette tips for transferring colonies
Sterile water
LB media with apropriate antibiotics

Procedure

Preparing the template DNA from the transformants:

Pick a number of transformants, typically 3-10, from each plate of interest and mark them on the back of the plate.
Set up 2 eppendorf tubes for each colony and mark them accordingly:
Fill the first one (1) with 15 µL MQ water.
The other one (2) remains empty for now.
Transfer each colony to the eppendorf containing 15 µL water using a sterile toothpick, inoculation loop or autoclaved pipette tip.
Transfer 5 µL of the water from (1) to the empty (2) tube. This tube (2) is now for safekeeping in case the colony PCR shows that the transformant in question contains the correct insertion.
Boil the (1) tubes for 10 minutes at 98 °C. Prepare the PCR mastermix, while the colonies are boiling.

Setting up the PCR itself:

Set up a 25 µL reaction for each colony to be screened, as per NEB'S one-taq PCR protocol (see the protocol for troubleshooting).

Component	25 µL reaction
5x OneTaq Standard Reaction Buffer	5 µL
10 mM dNTP	0,5 µL
10 µM Forward Primer	0,5 µL
10 µM Reverse Primer	0,5 µL
OneTaq DNA Polymerase	0.125 µL
Template	1 µL from the (1) tube
Nuclease-Free Water	up to 25 µL

Run the PCR in the thermocycler (use this website to calculate temperatures used based upon the primers and polymerase used).
Run the products on a gel to check for correct insertion.
Prepare an O/N culture from the (2) tubes that have the correct insertions by adding 1 mL LB media to the tube, mixing it and transfer a W-tube containing 4 mL LB media.

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The procedure determine the glucose concentration of a sample using 3,5-dinitroalicylic acid (DNS). Adapted by Philip Sørensen and Jacob Mejlsted from D. Navarro et al. and Worthington biochem.

Materials

Plate reader

DNS reagent

1 gram of 3,5-dinitrosalicylic acid
Milli-Q water
30.0 grams sodium potassium tartrate tetrahydrate
20 mL 2 M NaOH

Consumables

Microtiter plate

Procedure

Preparation of DNS reagent

Dissolve 1 g of 3,5-dinitrosalicylic acid in 50 mL Milli-Q water
Add slowly 30.0 grams of sodium potassium tartrate tetrahydrate
Add 20 mL of 2 M NaOH
Dilute to a final volume of 100 mL with reagent grade water. Protect from carbon dioxide and store no longer than 2 weeks.

Running the assay

100 µL media/sample is added to the microtiter plate
100 µL DNS regeant is added to each sample
Plate is heated to 98 °C for 10 min
Absorbance at 540 nm is measured
Remember to include a standard to determine actual concentrations

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Adapted by Jacob Mejlsted from NEB's product information and Barrick Lab
This protocol describes digestion of DNA with DpnI. This can be used after a PCR reaction to remove the template DNA. It is recommended that the PCR product is purified before this digestion is conducted, but not necessary.

Materials

Chemicals

DpnI enzyme

Procedure

DpnI digestion

Add 1 µL of DpnI to finished 50 µL PCR reactions (or 0.5 µL to 25 µL reactions). Pipet or invert to mix.
Incubate the mixture at 37 °C for 1-2 hrs.
Alternatively, the solution can be left overnight at room temperature. Periodic mixing may aid digestion (but is unnecessary).
PCR cleanup or gel-purify the reaction for downstream processes OR heat inactivate at 80 °C for 20 min.

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Adapted by Jacob Mejlsted & Kyle Rothschild

Materials

Equipment

Autoclave
Freezer
Centrifuge
Spectrophotometer

Consumables

PCR tubes
50 mL falcon tubes

Chemicals

MgCl₂
CaCl₂
SOC/SOB
Glycerol

Procedure

Day 1:

Autoclave the following:

Minimum 200 mL 0.1 M MgCl₂
Minimum 150 mL 0.1 M CaCl₂
Minimum 100 mL 85 mM CaCl₂ + 15% glycerol (v/v)
Minimum 700 mL SOC
3 x shakeflasks (500 mL)

Freeze at -20 °C (after autoclaving)

0.1 M MgCl₂
0.1 M CaCl₂
85 mM CaCl₂ + 15% glycerol (v/v)
Minimum 10 x 50 mL falcon tubes
PCR tubes

Day 2:

Culture growth
Early in the morning, start cooling the Sorval centrifuge to 4 °C
Pour 200 mL SOB media into each of the shakeflask (one shakeflask per starter culture).
Mark the shakeflasks to match the startercultures
Measure the OD 600 of each starterculture, and inoculate the shakeflask with a volume so the final OD 600 value in the shakeflask culture becomes 0.01
Grow the shakeflask culture at 37 °C with shaking. Measure OD values of the sample every 20 minutes once the OD 600 value is above 0.2.
When OD 600 is between 0.3 and 0.4, put the cultures into an ice bath immediately, and swirl the shake flash around in the cold water to cool culture. Chill the culture in the ice water for 20-30 minutes, occasionally swirling the cultures.
FROM THIS STAGE ON, KEEP CELLS AT ICE/4 °C AT ALL TIMES
For each shakeflask culture, pour the culture into 3 x 50 mL frosted falcon tubes from the freezer.
Keep the tubes on ice
Centrifuge falcon tubes at 3000 x g for 15 min at 4 °C (Spin #1 of 4)
Discard supernatant, and resuspend cells in 15 mL icecold 0.1 M MgCl₂
Keep tubes with cells on ice
Pool the resuspended cells into one of their matching falcon tubes, so you now have 3 different 50 mL falcon tubes, one with cells corresponding to each of the starter cultures you had.
Keep tubes on ice
Centrifuge falcon tubes at 2000 x g for 15 min at 4 °C (Spin #2 of 4)
Discard the supernatant, and resuspend pellet in 40 mL icecold 0.1 M CaCl₂
Keep tubes on ice
Let cell suspensions stand in ice for 20-30 minutes
Centrifuge falcon tubes at 2000 x g for 15 min at 4 °C (Spin #3 of 4)
Discard supernatant, and resuspend pellet in 10 mL icecold 85 mM CaCl₂ + 15 % glycerol
Keep tubes on ice
Centrifuge falcon tubes at 1000 x g for 15 minutes at 4 °C (Spin #4 of 4)
Pellet might look small and will be a bit fragile. Handle tubes with care when taking them out of centrifuge
Attention: The next few steps are best done on ice inside a LAF bench
Resuspend pellet in 800 µL ice-cold 85 mM CaCl₂ + 15 % glycerol
Put falcon tubes on ice
Immediately after cells are confirmed resuspended, aliquot 30 µL of the competent cell culture into the chilled PCR tubes
Put tubes into -80 °C freezer as fast as possible

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Here, a description of the instruments, settings, and general protocol for the fluorescence measurements can be found.

Materials

SpectraMax iD3 or equivalent plate reader

Consumables

Corning® Clear Polystyrene 96-Well Microplate

Procedure

Fluorescence measurements

If not done already, 100 µL of sample was loaded into the well of a spectophotometry plate.
The RFP was measured using the following settings: (Ex/Em)

mCherry: 580/625
GFP: 488/530

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Protocol for PCR & Gibson adapted from the Gibson Assembly Cloning Guide 2nd edition
Protocol for template removal adapted from NEBcloner
Protocol for PCR Purification adapted from Qiagen QIAquick® PCR Purification Kit
Protocol for MiniPrep adapted from Qiagen's QIAprep® Spin Miniprep Kit

Procedure

PCR Amplfication of DNA Fragments

Prepare PCR reaction (see table)

Component	Volume
Insert or vector DNA (100 pg/µL - 1 ng/µL in TE)	0.5 µL
10 µM Forward Primer	2.5 µL
10 µM Reverse Primer	2.5 µL
10 mM dNTPs	1 µL
5X Phusion HF Buffer	10 µL
Phusion DNA Polymerase (2 U/µL)	0.5 µL
Nuclease-free Water	33 µL
Total	50 µL

Run reaction in a thermocycler

Step	Temperature	Duration	Number of Cycles
Initial denaturation	98 °C	30 seconds	1 cycle
Amplification	98 °C	10 seconds	25-30 cycles
	Primer Tm	20 seconds
	72 °C	30 seconds/kb
Final extension	72 °C	5 minutes	1 cycle
Hold	4 °C	-	1 cycle

Template Removal

Set up the reaction as follows:

Component	50 µL Reaction
Component	50 µL Reaction
DNA	1 µg
10X CutSmart Buffer	5 µL (1X)
DpnI	1.0 µL (or 10 units)
Nuclease-free Water	to 50 µL

Incubate at 37 °C for 5–15 minutes (DpnI is Time-Saver qualified).
Optional: Heat inactivate at 80 °C for 20 min if not doing a DNA purification step

PCR Purification

Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10 µL 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow
Place a QIAquick column in a provided 2 mL collection tube
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s. Discard flow-through and place the QIAquick column back in the same tube.
To wash, add 750 µL Buffer PE to the QIAquick column centrifuge for 30–60 s. Discard flow-through and place the QIAquick column back into the same tube.
Centrifuge the QIAquick column once more in the provided 2 mL collection tube for 1 min to remove residual wash buffer.
Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.
To elute DNA, add 50 µL Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µL elution buffer to the center of the QIAquick membrane, let the column stand for 1 min and then centrifuge.

Mid-way analysis

Add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
Run the gel and evaluate the results
NanoDrop the purified DNA to determine the concentration

Gibson Assembly using a HiFi 1-Step Kit

Thaw Gibson Assembly HiFi 1-step Master Mix on ice
Combine insert and vector DNA to a total volume of:
5 µL for the 2X kit or
7.5 µL for the HC 4X kit
Vortex the master mix
On ice, combine
5 µL DNA and 5 µL Master Mix for the 2X kit or
7.5 µL DNA and 2.5 µL Master Mix for the HC 4X kit
Mix well andbriefly centrifuge
Incubate at 50 °C for 1 hour
Store reactions at -20 °C or use in 1-2 µL per reaction for transformation

Transformation protocol

Thaw chemically-competent cells on ice
Add 2 µL of the chilled assembly product to the competent cells. Mix gently by pipetting up or down or by flicking the tube 4-5 times. Do NOT vortex
Place the mixture on ice for 30 minutes. Do not mix
Heat shock at 42 °C for 30 seconds. Do not mix
Transfer tubes to ice for 2 minutes
Add 950 µL of room temperature SOC media to the tubes
Incubate the tube for 37 °C for 60 minutes. shake vigerously (250 rpm) or rotate
Warm selection plates to 37 °C
Spread 100 µL of the cells onto the selection plates.
Note: Use Amp plates for the positive control
Incubate overnight at 37 °C.

Day 2:

Plate counting: Count CFU and control plates
Innoculate LB + antibiotic media for O/N culture

Day 3: Miniprep

Use a miniprep kit to purify the assembled plasmids.

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This protocol is made to assembly parts with TypeIIs enzyme BsaI HF v2. These parts will have the standard MoClo overhangs. Protocol adapted by Philip Sørensen from a Evry Paris-Saclay's protocol.

Materials

Reagens

T4 DNA ligase
10X T4 DNA ligase buffer
BsaI HF v2 enzyme (NEB)
Reciever plasmid
DNA fragments with BsaI overhangs
Milli-Q water

Materials

PCR tubes
10 µL pipette tips
Thermocycler

Procedure

Setting up the reaction

In a PCR tube, mix the following:
- 0.5 µL of T4 DNA Ligase
- 2 µL of 10X T4 DNA Ligase Buffer
- 0.5 µL of BsaI HF v2 restriction enzyme
- 100 ng of receiver plasmid
- Equimolar amounts of inserts
- Milli-Q for a total volume of 20 µL.
Mix gently
Place the tube on a thermocycler

Thermocycler program

Step	Temp	Time
Activation of BsaI HF v2	37 °C	5 min
Activation of T4 ligase	16 °C	5 min
Repeat step 1 & 2 for 25 cycles
Inactivation BsaI HF v2	65 °C	20 min
Inactivation T4 ligase	85 °C	10 min
Hold	4 °C	Hold

The cloned plasmids can then be transformed as described in the Hifi assembly and transformation protocol.

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This protocol is made to assembly parts with TypeIIs enzyme SapI. Protocol adapted by Philip Sørensen from a Evry Paris-Saclay's protocol.

Materials

Reagens

T4 DNA ligase
10X T4 DNA ligase buffer
SapI enzyme (NEB)
Reciever plasmid
DNA fragments with SapI overhangs
Milli-Q water

Reagens

PCR tubes
10 µL pipette tips
Thermocycler

Procedure

Setting up the reaction

In a PCR tube, mix the following:
- 0.5 µL of T4 DNA Ligase
- 2 µL of 10X T4 DNA Ligase Buffer
- 0.5 µL of Type IIS restriction enzyme
- 100 ng of receiver plasmid
- Equimolar amounts of inserts
- Milli-Q for a total volume of 20 µL.
Mix gently
Place the tube on a thermocycler

Thermocycler program

Step	Temp	Time
Activation of SapI	37 °C	5 min
Activation of T4 ligase	16 °C	5 min
Repeat step 1 & 2 for 25 cycles
Inactivation SapI	65 °C	20 min
Inactivation T4 ligase	85 °C	10 min
Hold	4 °C	Hold

The cloned plasmids can then be transformed as described in the Hifi assembly and transformation protocol.

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Adapted by Jacob Mejlsted & Joen Haahr Jensen from NEB's HiFi assembly protocol.

Materials

(X is the number of reactions)

Consumables

X PCR tubes for each reaction + 1 for positive control
X eppendorf tupe for each reaction + 1 for postive control
X selection plate for each reaction
1 Amp plate for positive control

Chemical

Hifi DNA assembly Master mix
Sterile Milli-Q water
Competent E. coli cells
Prepared DNA fragments for assembly

Procedure

Assembly protocol

Set up the following reaction on ice:
* If the inserts are less than 200 bp, use a 5 fold excess of inserts instead of a 2 fold excess
** If a greater number of fragments are assembled, increase the volume of the reaction and use additional HiFi DNA assembly master mix

	Recommended amount of fragments used for assembly
	2-3 Fragments*	4-6 Fragements**	Postive control
Recommended DNA Molar Ratio	Vector:insert = 1:2	Vector:insert = 1:1
Total amount of DNA fragments	0.03-0.3pmols X µL	0.2-0.5 pmols X µL	10 µL
NEB Hifi Assembly master mix	10 µL	10 µL	10 µL
Milli-Q water	10-X µL	10-x µL	0 µL
Total volume	20 µL	20 µL	20 µL

Incubate the reaction samples in a thermocycler at 50 °C for 15 minutes (when 2-3 fragments are assembled) or 60 minutes (when 4-6 fragments are assembled). Following incubation, store the reaction samples at -20 °C for subsequent transformation
Note: Extended incubation up to 60 minutes can in some cases improve transformation efficiency

Transformation protocol

Thaw chemically-competent cells on ice
Add 2 µL of the chilled assembly product to the competent cells. Mix gently by pipetting up or down or by flicking the tube 4-5 times. Do NOT vortex
Place the mixture on ice for 30 minutes. Do not mix
Heat shock at 42 °C for 30 seconds. Do not mix
Transfer tubes to ice for 2 minutes
Add 950 µL of room temperature SOC media to the tubes
Incubate the tube for 37 °C for 60 minutes. shake vigerously (250 rpm) or rotate
Warm selection plates to 37 °C
Spread 100µL of the cells onto the selection plates.
Note: Use Amp plates for the positive control
Incubate overnight at 37 °C.

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Protocol for production of 1 liter of minimal media

Materials

50 mL of 20% w/V D-glucose stock
50 mL of 20x nitrate salts stock
1 mL 1000x Trace elements stock
1 mL of 1% thiamine stock
20 g agar

Procedure

Media preparation

Mix ingredients in flask and add MiliQ water to 1 litre
Mix with magnet stirrer until the sugar is dissolved
Autoclave at 121 °C for 20 min

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Here is a list of miscellaneous protocols, all of which were performed according to the manufacturer's and/or the referenced specifications. .

Protocols

Gel electrophoresis

Gels contained 1% agarose and were stained with 1000X SYBR Safe (Invitrogen)
DNA fragment solutions were combined with 6X Purple Loading Dye (New England Biolabs).
Samples were run in 1X TAE running buffer at 100 V with a 1kb DNA ladder (New England Biolabs).
Electrophoresis was run on the RunOne Electrophoresis System (Embi Tec).
Pictures of the resulting gels were taken with the ChemiDoc™ XRS+ Imaging System or equivalent.

Gel purifications

Isolated DNA bands were purified via the QIAquick Gel Extraction kit (QIAGEN).

PCR spin purifications

When deemed necessary, plasmid DNA was spin purified, rather than gel purified, via the QIAquick PCR Purification Kit (QIAGEN).

Plasmid miniprep purification

Plasmids were purified using the QIAprep Spin Miniprep Kit (QIAGEN).

DNA sequencing

DNA was verified by Sanger sequencing, using the Eurofins’ overnight sanger sequencing service.

Primer/DNA resuspension

Primers were diluted to a stock concentration of 100 µM and a working concentration of 10 µM.
Synthetic DNA (e.g. synthetic promoters) were diluted to 25 ng/µL or 40 ng/µL, depending on downstream uses.

This protocol is for linearising backbones with the PacI+Nt.BbvCI USER casette. This will allow assembly of PCR product with USER tails to be assembled into this linearised plasmid.

Materials

For digest I

Plasmid: 40 µL
PacI: 2 µL
Cutsmart buffer (NEB): 10 µL
MQ water: 48 µL

For digest II

Finished digest I
Nt.BbvCI: 2 µL

Procedure

Digest I

MIx all the reagents as mentioned above, and incubate at 37 °C overnight

Digest II

Take the digest I reaction from the 37 °C incubator, add in the 2 µL Nt.BbvCI to the reaction mixture, and incubate for 2 hours at 37 °C
Heat inactivate the samples at 80 °C for 30 min

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Protocol for PCR using the Phusion polymerase adapted by Jacob Mejlsted from the NEB protocol for the same product.

Materials

DNA

Consumables

PCR tubes (1 per reaction + 1 for positive control)

Chemicals

Forward primers
Reverse primers
Nuclease-free water
Phusion DNA Polymerase
5X Phusion HF or GC Buffer
10 mM dNTPs
DMSO (optional)

Procedure

PCR Amplfication of DNA Fragments

Prepare PCR reaction (see table)

Component	20 µL Reaction	50 µL Reaction	Final Concentration
Nuclease-free water	to 20 µL	to 50 µL
5X Phusion HF or GC Buffer	4 µL	10 µL	1X
10 mM dNTPs	0.4 µL	1 µL	200 µM
10 µM Forward Primer	1 µL	2.5 µL	0.5 µM
10 µM Reverse Primer	1 µL	2.5 µL	0.5 µM
Template DNA	variable	variable	< 250 ng
DMSO (optional)	(0.6 µL)	(1.5 µL)	3%
Phusion DNA Polymerase	0.2 µL	0.5 µL	1.0 units/50 µL PCR

Alternatively, a master mix can be prepared

Reactant	Per reaction (50µL) [µL]	Mastermix [µL]
Number of reactions	1	10
5X Phusion HF or GC Buffer	10	100
10 mM dNTPs	1	10
10 µM Forward Primer	2.5	Added individually
10 µM Reverse Primer	2.5	Added individually
Template DNA	variable	Added individually
Phusion DNA Polymerase	0.5	5
DMSO (optional)	0	0
Milli-Q	33.5	335

Run reaction in a thermocycler
*15 seconds/kb works for most reactions. 30 seconds/kb can be used for more complex reaction, such as cDNA.

Step	Temperature	Duration	Number of Cycles
Initial denaturation	98 °C	30 seconds	1 cycle
Amplification	98 °C	10 seconds	25-30 cycles
	Primer Tm	20 seconds
	72 °C	15-30 seconds/kb*
Final extension	72 °C	5-10 minutes	1 cycle
Hold	4 °C	-	1 cycle

Click below for a lab friendly version of this protocol

Adapted by Jacob Mejlsted from the NEB protocol for the same product.

Materials

1 PCR tube per reaction

Chemicals

OneTaq standard reaction buffer
dNTPs
Primers
DNA template
OneTaq DNA Polymease

Procedure

Method

To a tube add the following

Component	25 μL reaction***	50 μL reaction***	Final Concentration
5X OneTaq Standard Reaction Buffer*	5 µL	10 μL	1X
10 mM dNTPs (#N0447)	0.5 µL	1 μL	200 µM
10 µM Forward Primer	0.5 µL	1 μL	0.2 µM
10 µM Reverse Primer	0.5 µL	1 μL	0.2 µM
OneTaq DNA Polymerase	0.125 µL	0.25 µL	1.25 units/50 µL PCR**
Template DNA**	variable	variable	< 1,000 ng
Nuclease-free water	to 25 µL	to 50 µL

*OneTaq GC Reaction Buffer and High GC Enhancer can be used for difficult amplicons
**For plasmids or viral DNA, use 1 pg–10 ng DNA for a 50 µL reaction.
***It can be advantagous to pool some of the parts into a master mix, as some labs cannot dispense 0.125 µL accurately.

Alternatively, a master mix can be prepared

Reactant	Per reaction (50µL) [µL]	Mastermix [µL]
Number of reactions	1	10
5X OneTaq Standard Reaction Buffer*	10	100
10 mM dNTPs (#N0447)	1	10
10 µM Forward Primer	1	Added individually
10 µM Reverse Primer	1	Added individually
OneTaq DNA Polymerase	0.25	2.5
Template DNA	1	Added individually

Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary.

STEP	TEMP	TIME
Initial Denaturation	94 °C	30 seconds
30 Cycles	94 °C	15-30 seconds
	45-68 °C	15-60 seconds
	68 °C	1 minute per kb
Final Extension	68 °C	5 minutes
Hold	4-10 °C

PCR products can then by digested by DpnI, purified, stored at -20 C, or a combination.

Click below for a lab friendly version of this protocol

Protocol for PCR using the Q5 2X master mix adapted by Jacob Mejlsted from the NEB protocol for the same product.

Materials

DNA

Consumables

PCR tubes (1 per reaction + 1 for positive control)

Chemicals

Forward primers
Reverse primers
Nuclease-free water
Q5 2X Master Mix

Procedure

PCR Amplfication of DNA Fragments

Prepare PCR reaction (see table)

Component	25 µL reaction	50 µL reaction
Q5 High-Fidelity 2X Master Mix	12.5 µL	25 µL
10 µM Forward Primer	1.25 µL	2.5 µL
10 µM Reverse Primer	1.25 µL	2.5 µL
Template DNA*	variable (0ften 0.5 µL)	variable (0ften 1 µL)
Nuclease-Free Water	to 25 µL	to 50 µL
Total	25 µL	50 µL

*Template DNA:
For a 50 µL reaction, 1 ng-1 µg is recommended for genomic DNA and 1 pg-10 ng is recommended for plasmid or viral DNA.

Run reaction in a thermocycler

Step	Temperature	Duration	Number of Cycles
Initial denaturation	98 °C	30 seconds	1 cycle
Amplification	98 °C	10 seconds	25-30 cycles
	Primer Tm	20 seconds
	72 °C	30 seconds/kb
Final extension	72 °C	2 minutes	1 cycle
Hold	4 °C	-	1 cycle

The PCR products can then be stored at -20 °C, used directly, or purified using PCR purification or gel extraction

Click below for a lab friendly version of this protocol

This protocol describes the required components and method for a X7 polymerase mediated PCR reaction. .

Materials

Consumables

PCR strip/tubes

Procedure

PCR Amplfication of DNA Fragments

Prepare PCR reaction (see table below)

COMPONENT	50 µL REACTION
CXL buffer	10 µL
10 mM dNTPs	1 µL
10 µM Forward Primer	2.5 µL
10 µM Reverse Primer	2.5 µL
Template DNA	Variable (often 1 µL)
Pfu X7 Polymerase	0.5 µL
DMSO	1.5 µL
Nuclease-Free Water	to 50 µL

Run reaction in a thermocycler

Thermocycler PCR program

Step	Temperature	Duration	Number of Cycles
Initial denaturation	95 °C	2 minutes	1 cycle
Amplification	95 °C	30 seconds	25-30 cycles
	Primer Tm	30 seconds
	72 °C	around 1 min/kb*
Final extension	72 °C	5 minutes	1 cycle
Hold	4 °C	-	1 cycle

The PCR products can then be stored at -20 °C, used directly, or purified using PCR purification or gel extraction.

Click below for a lab friendly version of this protocol

This protocol is an adaptation of a GUS (β-glucuranidase) assay provided by Zofia Dorota Jarczynska and adapted by Jacob Mejlsted.

Materials

Extraction buffer (1L)

50 mM Na₃PO₄, pH 7 (50 mL of 1 M stock solution)
10 mM β-mercaptoethanol (0.7 mL of 14.4 M stock solution)
10 mM Na₂EDTA (20 mL of 0.5 M Na₂EDTA stock solution)
0.1% Sodium Lauryl Saccosine (10 mL of 10% Sarcosyl)
0.1% Triton X-100 (10 mL f 10% Triton)
Milli-Q water up to 1L

Equipment

Centrifuge with cooling
Plate reader
Homogenizer
Beads (big, metal)
Sterile funnels with mira cloth

Chemicals

Bradford reagent (Like SigmaAldrich B6916)
BSA standard (Bovine Serum Albumin 2 mg/mL)

Procedure

Protein Extraction

CRITICAL Set the centrifuge at 4 °C
Seperate biomass from supernantant using mira cloth or other methods
Take ~100 mg biomass and place in a FastPrep tube with one big metal bead
Place the tubes in liquid nitrogen
Homogenize for 1 min at 45 Hz
Add 500 µL extraction buffer
Homogenize for 2 min at 45 Hz
Centrifuge for 2 min at 10,000 x g at 4 °C
Collect the liquid phase and place in a new tube
Centrifuge for 15 min at 10,000 x g at 4 °C
Aliquote the supernantant into new tubes
Store at -80 °C

Bradford Assay

Transfer 100 µL protein sample into a microtiter place
Measure fluorescense at the appripriate wavelenghts

Fluorescence Assay (optional)

Mix 20 µL protein sample with 230 µL Bradfor reagent
Transfer to a new microtiter plate
Measure the blue color at 595 nm
Remember to include a standard curve and Bradford reagent
Determine protein concentration

Click below for a lab friendly version of this protocol

Adapted by Jacob Mejlsted from iGEM's protocol

Materials

Resuspended DNA to be transformed
10 pg/µL Positive transformation control DNA (e.g. pSB1C3 w/ BBa_J04450, RFP on high-copy chloramphenicol resistant plasmid. Located in the Competent Cell Test Kit.)
Competent Cells (50 µL per sample)
1.5 mL Microtubes
SOC Media (950 µL per sample)
Petri plates w/ LB agar and antibiotic (2 per sample)

Equipment

Floating Foam Tube Rack
Ice & ice bucket
Lab Timer
42 °C water bath
37 °C incubator
Sterile spreader or glass beads
Microcentrifuge

Procedure

Method: Day 1

Resuspend DNA in selected wells in the Distribution Kit with 10 µL dH₂O. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
Label 1.5 mL tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5 mL tubes (one tube for each transformation, including your control) in a floating foam tube rack.
Thaw competent cells on ice: This may take 10-15 min for a 260 µL stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
Pipette 50 µL of competent cells into 1.5 mL tube: 50 µL in a 1.5 mL tube per transformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 1.5 mL tube for your control.
Pipette 1 µL of resuspended DNA into 1.5 mL tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
Pipette 1 µL of control DNA into 2 mL tube: Pipette 1 µL of 10 pg/µL control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
Close 1.5 mL tubes, incubate on ice for 30 min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
Heat shock tubes at 42 °C for 45 sec: 1.5 mL tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.
Incubate on ice for 5 min: Return transformation tubes to ice bucket.
Pipette 950 µL SOC media to each transformation: SOC should be stored at 4 °C, but can be warmed to room temperature before use. Check for contamination.
Incubate at 37 °C for 1 hours, shaking at 200-300 rpm
Pipette 100 µL of each transformation onto petri plates. Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
Incubate transformations overnight (14-18 hours) at 37 °C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.

Method: Day 2

Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.
Count colonies for control transformation: Count colonies on the 100 μL control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10⁸ to 6x10⁸ CFU/µg DNA.

Click below for a lab friendly version of this protocol

Here, the procedure for sampling of the shake flask and bioreactor cultivations is described.

Materials

Consumables

10 mL syringe (bioreactor)
15 mL falcon tube
1.5 mL eppendorf tube
Corning® Clear Polystyrene 96-Well Microplate

Procedure

Shake flask cultivation

In a clean bench, approximately 1 mL of sample containing biomass was sampled via pipetting.
100 µL sample containing biomass was transferred to a spectrophotometry plate.
200 µL of sample was spun down at 10.000 g for 3 min and 100 µL transferred to a spectrophotometry plate.
Immediately after sampling, samples in the spectrophotometry plate was measured for fluorescence according to the fluorescence measurement protocol, and the remainder of the sample in the eppendorf tubes stored at -20 °C.

Bioreactor cultivation

For sampling of the bioreactor, extra care was taken to prevent contamination.
The sterile 10 mL syringe was sprayed with 70% ethanol and inserted in the sampling tube.
The clamps on the sampling tube were loosened and 5 mL sample was drawn and discarded.
The syringe was again sprayed with 70% ethanol, and reinserted in the sampling tube.
Approximately 10 mL sample containing biomass was drawn and transferred to a 15 mL falcon tube.
The clamps on the sampling tube were tightened, and the sampling tube sprayed with 70% ethanol.
The samples were stored at -20 C for later protein purification.

Click below for a lab friendly version of this protocol

Here, we describe the protocol for the set-up and execution of the shake flask cultivations. Please, click here for the media recipe, here for sampling procedures for the specifics regarding sampling, and here for details regarding the fluorescence measurements.

Materials

Pyrex shake flasks (with baffles)
Minimal media
Spore suspension

Consumables

Cotton stopper
Tin foil

Procedure

Preparations and inoculation

A breathable cotton ball was added to the mouth of a glass shake flask (with baffles), covered with tin foil, and then autoclaved.
150 mL minimal media was added to each shake flask.
The shake flasks were inoculated with their respective spore solutions to a final concentration of 10⁶ spores/mL.
Shake flasks were incubated for 5 days as described below.

Conditions

Shake flasks were incubated at 30 °C at 150 rpm for 5 days.
Samples were taken as regularly as described in sampling procedures.

Click below for a lab friendly version of this protocol

The following is a quick and easy protocol for creating the spore suspensions used for storage and the inoculation of the shake flask, biolector, and bioreactor cultivations. .

Materials

Fully grown fungi plates (with spores)
Drigalski spatula
96% ethanol
FireBoy or other flame-sterilizing equipment
Sterile Milli-Q water
Sterile mira cloth
Sterile falcon tubes
P1000 pipette and tips
Scissors
Microscope
Counting chamber

Procedure

Creating the spore suspensions

Add mira cloth to a falcon tube.
Pour 2-3 mL of Milli-Q water onto a plate
Clean and sterilize a drigalski spatula using ethanol and fire.
Let the drigalski cool off in the water before commencing the scraping. Then, carefully scrape off the spores from the plate. This should be done with the lid still on and VERY careully as the spores are hydrophobic and would much rather just fly around than being suspended into the water.
When all of the spores have been suspended into the water, transfer the liquid to the mira cloth using a pipette with the tip cut off.
When this is done, count the spores in the microscope. Usually, a 1000x dilution is necessary.
The spore suspension can be kept for up to 3 months at 4 °C.

Click below for a lab friendly version of this protocol

Protocol for production of 1 liter of fungal transformation media

Materials

342.30 g sucrose
50.0 mL Nitrate salts
1 mL Trace metal elements
1 mL Thiamine
21 g Bacto agar

Procedure

Mix ingredients in flask and add MiliQ water to 1 litre.
Mix with magnet stirrer until the sugar is dissolved.
Autoclave at 121 °C for 20 min.

Click below for a lab friendly version of this protocol

The following describes the USER-cloning protocol used during our project.

Materials

Reagents

USER-linearized insert(s)
USER-linearized backbone
USER enzyme
CutSmart buffer
Milli-Q water

Consumables

PCR-strip/tubes

Procedure

Preparing USER-reaction

In a PCR-tube, combine;
- 200 ng USER-linearized insert(s)
- 100 ng USER-linearized vector backbone
- 1 µL 10X CutSmart buffer
- 1 µL USER enzyme
- H2O to 10 µL.
Incubate reaction mix as described below.

Incubating reaction mix

In a PCR-machine, incubate as follows.

37 °C for 25 min
25 °C for 10 min
20 °C for 10 min
15 °CC for 10 min
10 °C until further use

Transform reaction mixture in E. coli as described in the Hifi assembly protocol

Click below for a lab friendly version of this protocol

The logos of our three biggest supporters, DTU Blue Dot, Novo Nordisk fonden and Otto Mønsted fonden

The logos of all of our sponsors, DTU, BioNordica, Eurofins Genomics, Qiagen, NEB New England biolabs, IDT Integrated DNA technologies and Twist bioscience

@@ Line 94: / Line 94: @@
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h4 class="media heading">Procedure</h4>
@@ Line 114: / Line 114: @@
 <div class="interlabspace">
-<H2 style="text-align: left;margin-bottom:20px;">Protocols</H2>
+<H2 style="text-align: left;margin-bottom:0px !important;">Protocols</H2>
+<p style="text-align: left;margin-bottom:20px !important;">Lab friendly downloads available for all protocols</p>
 <!--#############################################################################################
 Aspergillus niger protoplast protocol
 ##############################################################################################-->
-<button id="resuspensionofdna" class="collapsible">Aspergillus niger protoplast</button>
+<button id="protoplast" class="collapsible"><i>Aspergillus niger</i> protoplast</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 125: / Line 126: @@
 <p>
-This protocol is the adapted protocol from 223.
+This protocol is adapted from a protocol used by DTU Bioengineering.
 </p>
@@ Line 138: / Line 139: @@
 	<li>	Fungal plates (either streaked or 3-point)	</li>
 <li>	Drigalski spatula	</li>
-<li>	500 ml shake flasks	</li>
+<li>	500 mL shake flasks	</li>
 <li>	Counting chamber	</li>
-<li>	Solutions (APB, ATP, PCT, milli-Q, TM)	</li>
+<li>	Solutions (APB, ATP, PCT, Milli-Q, TM)	</li>
 <li>	30 °C incubator with shaking	</li>
-<li>	sterile tea spoon	</li>
+<li>	Sterile tea spoon	</li>
-<li>	mira cloth in funnel (sterile)	</li>
+<li>	Mira cloth in funnel (sterile)	</li>
 <li>	Glucanex	</li>
-<li>	magnet stirrer	</li>
+<li>	Magnet stirrer	</li>
-<li>	magnets	</li>
+<li>	Magnets	</li>
-<li>	50 ml sterile falcon tubes	</li>
+<li>	50 mL sterile falcon tubes	</li>
 <li>	0.45 µm filters	</li>
-<li>	50 ml syringe	</li>
+<li>	50 mL syringe	</li>
-<li>	centrifuge for falcon tubes	</li>
+<li>	Centrifuge for falcon tubes	</li>
 </ul>
@@ Line 159: / Line 160: @@
 	<li>	Aspergillus transformation buffer (ATB)	</li>
 <li>	Aspergillus protoplastation buffer (APB)	</li>
-<li>	500 ml shake flasks	</li>
+<li>	500 mL shake flasks	</li>
 <li>	YPD media	</li>
 </ul>
@@ Line 176: / Line 177: @@
 <h4>Day 1 (inoculation)</h4>
 <ul  start="3" class="protocolli">
-	<li>Add 95 ml of YPD media supplemented with uridine to a shake flask and transfer 5 ml of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 ml. It is a good idea to make more than one shake flask at a time.</li>
+	<li>Add 95 mL of YPD media supplemented with uridine to a shake flask and transfer 5 mL of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 mL. It is a good idea to make more than one shake flask at a time.</li>
 	<li>Incubate shake flasks at 30 °C, 150 RMP for 48 h.</li>
@@ Line 192: / Line 193: @@
 <h4> Protoplastation </h4>
 <ul  start="7" class="protocolli">
-	<li>Add glucanex to APB to get a final concentration of 40 mg glucanex per ml APB and dissolve glucanex via gentle magnetic stirring and no heat.</li>
+	<li>Add glucanex to APB to get a final concentration of 40 mg glucanex per mL APB and dissolve glucanex via gentle magnetic stirring and no heat.</li>
-	<li>Sterile filter 20 ml of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 ml syringe (there is a bit of resistance in the filter but that's ok).</li>
+	<li>Sterile filter 20 mL of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 mL syringe (there is a bit of resistance in the filter but that's ok).</li>
-	<li>Shake/incubate enzyme-mycelium mix at 30 °C, 150 RPM for 2-3 h.</li>
+	<li>Shake/incubate enzyme-mycelium mix at 30 °C, 150 rpm for 2-3 h.</li>
 <li>From now on, whenever you pipette anything with the cells in it, cut of the edge of the pipette tip and CAREFULLY(!) pipette the cells. If you don't do this, they break as they don't have a cell wall to keep them stable.</li>
-<li>Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/ml). Approved protoplast solutions are then diluted by pouring APB up to 40 ml. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile milli-Q H2O and carefully place 5 ml of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)</li>
+<li>Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/mL). Approved protoplast solutions are then diluted by pouring APB up to 40 mL. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile Milli-Q water and carefully place 5 mL of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)</li>
-<li>In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 ml and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.</li>
+<li>In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 mL and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.</li>
 <li>Count protoplasts in microscope by diluting a small sample 1:100.</li>
-<li>Resuspend protoplasts in 4 ml ATB to obtain concentrated solution.</li>
+<li>Resuspend protoplasts in 4 mL ATB to obtain concentrated solution.</li>
 </ul>
 </div>
@@ Line 205: / Line 206: @@
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/3/3e/T--DTU-Denmark--Aspergillus_protoplast_protocol.pdf
+" download="Aspergillus-protoplast-protocol.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
@@ Line 222: / Line 222: @@
 . Aspergillus Transformation
 ##############################################################################################-->
-<button id="resuspensionofdna" class="collapsible">Aspergillus Transformation</button>
+<button id="AspergillusTransformation" class="collapsible"><i>Aspergillus</i> transformation</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 228: / Line 228: @@
 <p>
-A quickoverview of the two different transformation protocols
+A quick overview of the two different transformation protocols
 </p>
@@ Line 251: / Line 251: @@
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
 <h4>Regular protocol</h4>
 <ul class="protocolli">
-	<li>	Add at MOST 20 uL DNA ( or max 25% of protoplast volume) and 100 uL of protoplasts in a 50 mL falcon tube.	</li>
+	<li>	Add at MOST 20 µL DNA ( or max 25% of protoplast volume) and 100 µL of protoplasts in a 50 mL falcon tube.	</li>
 <li>	Incubate in the falcon tube at RT for at least 30 min.	</li>
 <li>	Add 1mL of PCT.	</li>
@@ Line 270: / Line 270: @@
 <h4>Quick protocol</h4>
 <ul  start="10" class="protocolli">
-	<li>	Add at MOST 25 uL (1500-5000 ng) DNA ( or max 25% of protoplast volume) and 100 uL of protoplasts in a 2 ml Eppendorf tube. 1 uL (100 ng) pac6 as positive control.	</li>
+	<li>	Add at MOST 25 µL (1500-5000 ng) DNA ( or max 25% of protoplast volume) and 100 µL of protoplasts in a 2 mL Eppendorf tube. 1 µL (100 ng) pac6 as positive control.	</li>
 <li>	Add 150 μL PCT with large-nozzle pipette tip.	</li>
 <li>	Gently mix by swirling – careful the protoplasts are fragile.	</li>
@@ Line 281: / Line 281: @@
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/3/3b/T--DTU-Denmark--Aspergillus_Transformation_protocols.pdf
+" download="Aspergillus-transformation-protocol.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
@@ Line 294: / Line 294: @@
 Bacterial Glycerol Stock
 ##############################################################################################-->
-<button id="proto3aassembly" class="collapsible">Bacterial Glycerol Stock</button>
+<button id="proto3aassembly" class="collapsible">Bacterial glycerol stock</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 300: / Line 300: @@
 <p>
-Protocol for producing a glycerol stock of E. coli adapted by Jacob Mejlsted from <a href="https://www.addgene.org/protocols/create-glycerol-stock/" target="_blank">Addgene's protocol</a>.</i>
+Protocol for producing a glycerol stock of <i>E. coli</i> adapted by Jacob Mejlsted from <a href="https://www.addgene.org/protocols/create-glycerol-stock/" target="_blank">Addgene's protocol</a>.</i>
 </p>
 </div>
-<div class="col-xs-4">
+<div class="col-sm-4 col-xs-12">
 <h3 class="media heading">Materials</h3>
@@ Line 321: / Line 321: @@
 </div>
-<div class="col-xs-8">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
@@ Line 327: / Line 327: @@
 <ul>
 	<li>After having cultivated a culture overnight, add 500 µL culture to 500 µL 50% glycerol in the selected tube</li>
-	<li>The stock can now be frozen at -80°C<br> If the stock is repeatedly thawed and frozen, it will reduce the shelf life of the culture</li>
+	<li>The stock can now be frozen at -80 °C<br> If the stock is repeatedly thawed and frozen, it will reduce the shelf life of the culture</li>
 </ul>
@@ Line 338: / Line 338: @@
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/b/bf/T--DTU-Denmark--Glycerol_stock.pdf
+" download="Bacterial-glycerol-stock.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
@@ Line 358: / Line 358: @@
 <p>
-This guide is to use the biolector. <br> NB! The machine is very slow - be patient!
+How to load and clean biolector plate.
 </p>
@@ Line 369: / Line 369: @@
 <ul class="protocolli">
-	<li>	biolector</li>
+	<li>Biolector</li>
+	<li>Minimal media</li>
+	<li>P1000 pipette and tips</li>
+	<li>P10 pipette and tips</li>
+	<li>Spore suspensions</li>
+	<li>Biolector plate</li>
+	<li>Lid for the biolector plate</li>
 </ul>
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
 <ul class="protocolli">
-	<li>	Turn on the machine (bottom in the back, to the right)	</li>
+        <li><b>NB! Make sure to do all work in a clean LAF-bench!</b></li>
-<li>	Principal window ----> “Monitor”	</li>
+        <li>Clean plate with ethanol and a cotton swab and leave to dry</li>
-<li>	When the lid opens, place the plate in the machine between the metal barrages - it should be firmly secured.	</li>
+        <li>Clean additionally using UV for >30 min.</li>
-<li>	For create a new program go to second window “Start …” and follow the steps:	</li>
+        <li>Add 1500 µl minimal media (+supplements if necessary) to each well.</li>
-<li>	Choose new protocol and give it a name (if it is a warm-up program, specify it - the machine needs to warm up for 20-30 min before you add the plate) ----> Next. Names should be written as DDMMYYiGEM_*temperature*_XXXXX	</li>
+        <li>Add spores to the wells to achieve a final concentration of 10<sup>6</sup> or 10<sup>7</sup> spores per mL.</li>
-<li>	Select the type of plate (MTP 48 wells FlowerPlate) ----> Next	</li>
+        <li>Add a lid for the plate. These are sticky and should be breathable, i.e. the white, kind of fabric-y lids</li>
-<li>	Choose Layout (48 MTP) ----> Next	</li>
+        <li>Run Biolector for 36 hours</li>
-<li>	Select rpm (1000 rpm), temperature and humidity control, and manual (by defect) ----> Next	</li>
+	<li>After run, fill all wells with ethanol and clean using "soft" things such as a pipette tip to get to the corners and a cue tip to clean the sides. Repeat this process until the plate is clean.</li>
-<li>	Select the filters to use (Biomass –> Gain 10, PFP –> Gain ?, GFP –> Gain ? ) ----> Next	</li>
-<li>	Choose cycle time (30 min for the warm-up program and 3 min (more if several filters are addded) for our program) ----> Start	</li>
-<li>	The machine will ask for refilling the deposit with water. Once done, press OK and the program will start running. Be sure to fill up water once a day.	</li>
-<li>	When the running is finished, press Stop and save the file in the computer (in the machine, it is saved automatically). You need to press the 'update' button on the computer to get the updated data.	</li>
-<li>	In the software go to “Data Management” and select “Transfer the data…”. You can create a new folder or select one already created and press create file. The file will be saved in the folder in cvs format.	</li>
 </ul>
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/1/1f/T--DTU-Denmark--Biolector.pdf
+" download="Biolector.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
@@ Line 406: / Line 406: @@
+<!--#############################################################################################
+Bioreactor cultivation
+##############################################################################################-->
+<button id="Bioreactorcultivation" class="collapsible">Bioreactor cultivation</button>
+<div class="excontent">
+<div class="row propadding">
+<div class="col-xs-12">
+<p>
+Protocol for preparation and execution of the 1 L bioreactor (Sartorius Stedim 1L) cultivations. Adapted from DTU course material by Helena Utzon.
+</p>
+</div>
+<div class="col-sm-4 col-xs-12">
+<h3 class="media heading">Materials</h3>
+<h4> Materials </h4>
+<ul class="protocolli">
+	<li> 1 L Sartorius Stedim 1L bioreactor </li>
+	<li> Sartorius Stedim BIOSTAT Q Plus control tower </li>
+<li> Off-gas </li>
+	<li> Minimal media </li>
+	<li> Sigma 204 Antifoam </li>
+	<li> Sterile arginine solution </li>
+	<li> Acid (2 M Sulfuric acid) </li>
+<li> Base (2 M Potassium hydroxide) </li>
+<li> Spore suspensions </li>
+</ul>
+<h4> Consumables </h4>
+<ul class="protocolli">
+	<li> 1 mL syringes </li>
+	<li> Lab grease </li>
+	<li> White strips </li>
+</ul>
+</div>
+<div class="col-sm-8 col-xs-12 protoco1">
+<h3 class="media heading">Procedure</h3>
+<h4> Assembly of the bioreactors </h4>
+<ul>
+	<li> Mount the inoculation, sampling, and temperature port from the bottom of the lid and secure them with the bolt from the top of the lid. </li>
+	<li> Mount the impellers at the correct height. </li>
+	<li> Attach the sparger and baffles, and secure them with the bolt at the top of the bioreactor. </li>
+	<li> Mount the condenser, preferably as close to the control tower as possible. </li>
+	<li> The mouth of the condenser is loosely packed with a bit of glass wool, and the condenser is closed off. </li>
+	<li> The temperature and sampling pipe is mounted, and the sampling tubing is attached and sealed off with both a butterfly clamp and a regular clamp. </li>
+	<li> The inoculation port (with membrane) is assembled and mounted.  </li>
+	<li> The 4-way connector is attached to the bioreactor, and the un-needed connections are closed off with a knotted piece of tubing. </li>
+	<li> Marprene tube is attached to the air inlet, and an air filter is attached to the tubing in the right direction.  </li>
+	<li> Assemble the bioreactor, using a bit of lab grease on the rubber gaskets. </li>
+	<li> Connect the empty 250 mL Blue Cap bottles (acid/base) to the 4-way connector, with tubing appropriate for the control tower pumps.  </li>
+	<li> Clamp tubing between the bioreactor and bottles, as close to the reactor as possible. Also, attach a clamp the tube leading to the air filter. </li>
+	<li> Secure all tubing with white strips. </li>
+	<li> Fill the reactor with 1 L of minimal medium (<a href="#Minimal-media" >see media recipe</a>). </li>
+	<li> Calibrate the pH-electrode, and pressure test the reactor. </li>
+	<li> Cover all open ends and filters with tin foil to protect them during autoclaving. </li>
+	<li> Check all clamps prior to autoclavation, and loosen the main screw to prevent overpressure. </li>
+	<li> Autoclave the bioreactor. </li>
+	<li> After autoclavation, close the loose fitting and mount the air supply. Set the flow rate on the DCU to 0.75 vvm, and remove all clamps except for on the sampling tubing. </li>
+	<li> Once the medium has cooled down, connect the condenser tubing, the temperature jacket tubing, pH-electrode, DO-electrode, and the stirrer. </li>
+	<li> Set the stirrer to 800 rpm and check the accuracy of the pH-electrode. Calibrate if needed. </li>
+	<li> Mount the tubing for the acid and base on their respective pumps, run the acid/base through the tubing and switch to “auto”. </li>
+<li> Calculate the inoculum volume to a final concentration of 10<sup>6</sup> spores/mL, and inoculate the bioreactors with the previously prepared spore suspensions (<a href="#Sporesuspensions" >See Here</a>). </li>
+<li> Add 50 µL of Sigma 204 Antifoam to the reactor. </li>
+<li> Due to species limitations, 0.7 g of arginine was added to the media of the positive control. </li>
+<li> Set the timer to zero at the DCU, and start the batch fermentation at the desired process values. </li>
+<li> Please find the ramps and process conditions for our bioreactor cultivations below. </li>
+</ul>
+<h4> Bioreactor conditions </h4>
+<ul start="3">
+<style type="text/css">
+.tg  {border-collapse:collapse;border-spacing:0;border:none;}
+.tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:0px;overflow:hidden;word-break:normal;}
+.tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:0px;overflow:hidden;word-break:normal;}
+.tg .tg-il2e{font-weight:bold;background-color:#09314f;color:#ffffff;text-align:left;vertical-align:top}
+.tg .tg-kftd{background-color:#efefef;text-align:left;vertical-align:top}
+.tg .tg-0lax{text-align:left;vertical-align:top}
+</style>
+<table class="tg">
+  <tr>
+    <th class="tg-il2e" colspan="2">Process conditions</th>
+  </tr>
+  <tr>
+    <td class="tg-0lax" colspan="2">Airflow inlet</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">Time (min)</td>
+    <td class="tg-kftd">ValueVVM (L/L/M)</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax">0</td>
+    <td class="tg-0lax">0.1</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">300</td>
+    <td class="tg-kftd">0.16</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax">420</td>
+    <td class="tg-0lax">0.4</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">600</td>
+    <td class="tg-kftd">1</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax" colspan="2">pH</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">Time (min)</td>
+    <td class="tg-kftd">ValuepH</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax">0</td>
+    <td class="tg-0lax">3</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">600</td>
+    <td class="tg-kftd">5</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax" colspan="2">Stirrer speed</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">Time (min)</td>
+    <td class="tg-kftd">ValueRPM</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax">0</td>
+    <td class="tg-0lax">100</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">300</td>
+    <td class="tg-kftd">200</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax">420</td>
+    <td class="tg-0lax">300</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">600</td>
+    <td class="tg-kftd">500</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax">720</td>
+    <td class="tg-0lax">800</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd" colspan="2">Temperature</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax">Time (min)</td>
+    <td class="tg-0lax">Value(°C)</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">Start-end</td>
+    <td class="tg-kftd">30</td>
+  </tr>
+</table>
+<li>Sampling can then be performed as described in <a href="#Samplingprocedures">the sampling protocol</a>
+</ul>
+</div>
+<h3> Click below for a lab friendly version of this protocol</h3>
+<a href="https://static.igem.org/mediawiki/2019/e/eb/T--DTU-Denmark--Bioreactor.pdf" download="Bioreactor-protocol.pdf">
+    <button class="butn protocolbtn">Download File</button>
+</a>
+</div>
+</div>
@@ Line 434: / Line 616: @@
 <li>	Transformants from whatever assembly method you fashion	</li>
 <li>	Eppendorf tubes	</li>
-<li>	Steril toothpicks/inoculation lops/pipette tips for transferring colonies	</li>
+<li>	Sterile toothpicks/inoculation lops/pipette tips for transferring colonies	</li>
 <li>	Sterile water	</li>
 <li>	LB media with apropriate antibiotics	</li>
@@ Line 442: / Line 624: @@
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
@@ Line 449: / Line 631: @@
 	<li>	Pick a number of transformants, typically 3-10, from each plate of interest and mark them on the back of the plate.	</li>
 <li>	Set up 2 eppendorf tubes for each colony and mark them accordingly:	</li>
-<li>	Fill the first one (1) with 15 µl MQ water.	</li>
+<li>	Fill the first one (1) with 15 µL MQ water.	</li>
 <li>	The other one (2) remains empty for now.	</li>
-<li>	Transfer each colony to the eppendorf containing 15 µl water using a sterile toothpick, inoculation loop or autoclaved pipette tip.	</li>
+<li>	Transfer each colony to the eppendorf containing 15 µL water using a sterile toothpick, inoculation loop or autoclaved pipette tip.	</li>
-<li>	Transfer 5 µl of the water from (1) to the empty (2) tube. This tube (2) is now for safekeeping in case the colony PCR shows that the transformant in question contains the correct insertion.	</li>
+<li>	Transfer 5 µL of the water from (1) to the empty (2) tube. This tube (2) is now for safekeeping in case the colony PCR shows that the transformant in question contains the correct insertion.	</li>
 <li>	Boil the (1) tubes for 10 minutes at 98 °C. Prepare the PCR mastermix, while the colonies are boiling.	</li>
@@ Line 459: / Line 641: @@
 <h4>Setting up the PCR itself:</h4>
 <ul  start="8" class="protocolli">
-	<li>Set up a 25 µl reaction for each colony to be screened, as per NEB'S one-taq PCR protocol (see the <a href="https://international.neb.com/protocols/2012/10/11/onetaqdnapolymerasem0480" target="_blank">protocol</a>for troubleshooting).</li>
+	<li>Set up a 25 µL reaction for each colony to be screened, as per NEB'S one-taq PCR protocol (see the <a href="https://international.neb.com/protocols/2012/10/11/onetaqdnapolymerasem0480" target="_blank">protocol</a> for troubleshooting).</li>
@@ Line 477: / Line 659: @@
    <tr>
      <th class="tg-nzhz">Component</th>
-     <th class="tg-nzhz">25 µl reaction</th>
+     <th class="tg-nzhz">25 µL reaction</th>
    </tr>
    <tr>
      <td class="tg-73oq">5x OneTaq Standard Reaction Buffer</td>
-     <td class="tg-vqzh">5 µl</td>
+     <td class="tg-vqzh">5 µL</td>
    </tr>
    <tr>
      <td class="tg-vfn0">10 mM dNTP</td>
-     <td class="tg-rbwf">0,5 µl</td>
+     <td class="tg-rbwf">0,5 µL</td>
    </tr>
    <tr>
      <td class="tg-73oq">10 µM Forward Primer</td>
-     <td class="tg-vqzh">0,5 µl</td>
+     <td class="tg-vqzh">0,5 µL</td>
    </tr>
    <tr>
      <td class="tg-vfn0">10 µM Reverse Primer</td>
-     <td class="tg-rbwf">0,5 µl</td>
+     <td class="tg-rbwf">0,5 µL</td>
    </tr>
    <tr>
      <td class="tg-73oq">OneTaq DNA Polymerase</td>
-     <td class="tg-vqzh">0.125 µl</td>
+     <td class="tg-vqzh">0.125 µL</td>
    </tr>
    <tr>
      <td class="tg-vfn0">Template</td>
-     <td class="tg-rbwf">1 µl from the (1) tube</td>
+     <td class="tg-rbwf">1 µL from the (1) tube</td>
    </tr>
    <tr>
      <td class="tg-73oq">Nuclease-Free Water</td>
-     <td class="tg-vqzh">up to 25 µl</td>
+     <td class="tg-vqzh">up to 25 µL</td>
    </tr>
 </table>
@@ Line 513: / Line 695: @@
 	<li>	Run the PCR in the thermocycler (use <a href="https://tmcalculator.neb.com/#!/main" target="_blank">this website</a> to calculate temperatures used based upon the primers and polymerase used).	</li>
 <li>	Run the products on a gel to check for correct insertion.	</li>
-<li>	Prepare an O/N culture from the (2) tubes that have the correct insertions by adding 1 ml LB media to the tube, mixing it and transfer a W-tube containing 4 ml LB media.	</li>
+<li>	Prepare an O/N culture from the (2) tubes that have the correct insertions by adding 1 mL LB media to the tube, mixing it and transfer a W-tube containing 4 mL LB media.	</li>
 </ul>
@@ Line 519: / Line 701: @@
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/4/41/T--DTU-Denmark--Colony_PCR.pdf" download="Colony-PCR.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
@@ Line 528: / Line 709: @@
+<!--#############################################################################################
+DNS Glucose Assay
+##############################################################################################-->
+<button id="DNS" class="collapsible">DNS glucose assay</button>
+<div class="excontent">
+<div class="row propadding">
+<div class="col-xs-12">
+<p>
+The procedure determine the glucose concentration of a sample using 3,5-dinitroalicylic acid (DNS).
+Adapted by Philip Sørensen and Jacob Mejlsted from <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2919459/" target="_blank">D. Navarro et al.</a> and <a href="http://www.worthington-biochem.com/AA/assay.html" target="_blank">Worthington biochem.</a>
+</p>
+</div>
+<div class="col-sm-4 col-xs-12">
+<h3 class="media heading">Materials</h3>
+<h4>Materials </h4>
+<ul class="protocolli">
+	<li>Plate reader</li>
+</ul>
+<h4>DNS reagent</h4>
+<ul class="protocolli">
+	<li>1 gram of 3,5-dinitrosalicylic acid</li>
+	<li>Milli-Q water</li>
+	<li>30.0 grams sodium potassium tartrate tetrahydrate</li>
+	<li>20 mL 2 M NaOH</li>
+</ul>
+<h4>Consumables </h4>
+<ul class="protocolli">
+	<li>Microtiter plate</li>
+</ul>
+</div>
+<div class="col-sm-8 col-xs-12 protoco1">
+<h3 class="media heading">Procedure</h3>
+<h4>Preparation of DNS reagent</h4>
+<ul>
+	<li>Dissolve 1 g of 3,5-dinitrosalicylic acid in 50 mL Milli-Q water</li>
+	<li>Add slowly 30.0 grams of sodium potassium tartrate tetrahydrate </li>
+<li>Add 20 mL of 2 M NaOH</li>
+	<li>Dilute to a final volume of 100 mL with reagent grade water. Protect from carbon dioxide and store no longer than 2 weeks.</li>
+</ul>
+<h4>Running the assay</h4>
+<ul start="3">
+	<li>100 µL media/sample is added to the microtiter plate</li>
+	<li>100 µL DNS regeant is added to each sample</li>
+	<li>Plate is heated to 98 °C for 10 min</li>
+<li>Absorbance at 540 nm is measured</li>
+	<li><b>Remember to include a standard to determine actual concentrations</b></li>
+</ul>
+</div>
+<h3> Click below for a lab friendly version of this protocol</h3>
+<a href="https://static.igem.org/mediawiki/2019/0/04/T--DTU-Denmark--DNS_assay.pdf" download="DNS-assay.pdf">
+    <button class="butn protocolbtn">Download File</button>
+</a>
+</div>
+</div>
@@ Line 535: / Line 781: @@
 DpnI Digestion of PCR Products
 ##############################################################################################-->
-<button id="DpnIDigestion" class="collapsible">DpnI Digestion of PCR Products</button>
+<button id="DpnIDigestion" class="collapsible">DpnI digestion of PCR products</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 560: / Line 806: @@
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
 <h4>DpnI digestion</h4>
 <ul class="protocolli">
-	<li>Add 1µL of DpnI to finished 50µL PCR reactions (or .5µL to 25µL reactions). Pipet or invert to mix.</li>
+	<li>Add 1 µL of DpnI to finished 50 µL PCR reactions (or 0.5 µL to 25 µL reactions). Pipet or invert to mix.</li>
 	<li>Incubate the mixture at 37 °C for 1-2 hrs. <br> Alternatively, the solution can be left overnight at room temperature. Periodic mixing may aid digestion (but is unnecessary).</li>
 		<li>PCR cleanup or gel-purify the reaction for downstream processes OR heat inactivate at 80 °C for 20 min.</li>
@@ Line 571: / Line 817: @@
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/6/69/T--DTU-Denmark--DpnI_digest.pdf" download="DpnI-digestion.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
@@ Line 588: / Line 833: @@
 E.coli Competent Cell
 ##############################################################################################-->
-<button id="CompetentCell" class="collapsible">E.coli Competent Cell</button>
+<button id="CompetentCell" class="collapsible"><i>E. coli</i> competent cell</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 594: / Line 839: @@
 <p>
-Adapted by Jacob Mejlsted & Kyle Rothschild from XXXX (ask Kyle)
+Adapted by Jacob Mejlsted & Kyle Rothschild
 </p>
@@ Line 608: / Line 853: @@
 <li>	Freezer	</li>
 <li>	Centrifuge	</li>
-<li>	Spektrophotometer	</li>
+<li>	Spectrophotometer	</li>
 </ul>
@@ Line 631: / Line 876: @@
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
@@ Line 640: / Line 885: @@
 	<li>	Minimum 200 mL 0.1 M MgCl<sub>2</sub>	</li>
 <li>	Minimum 150 mL 0.1 M CaCl<sub>2</sub>	</li>
-<li>	Minimum 100 mL 85 mM CaCl2 + 15% glycerol (v/v)	</li>
+<li>	Minimum 100 mL 85 mM CaCl<sub>2</sub> + 15% glycerol (v/v)	</li>
 <li>	Minimum 700 mL SOC	</li>
 <li>	3 x shakeflasks (500 mL)	</li>
 </ul>
-	<li>Freeze at -20 degC (after autoclaving)</li>
+	<li>Freeze at -20 °C (after autoclaving)</li>
 		<ul class="protocolli">
 	<li>	0.1 M MgCl<sub>2</sub>	</li>
@@ Line 659: / Line 904: @@
 <ul  start="3" class="protocolli">
 	<li>	Culture growth	</li>
-<li>	Early in the morning, start cooling the Sorval centrifuge to 4 C	</li>
+<li>	Early in the morning, start cooling the Sorval centrifuge to 4 °C	</li>
 <li>	Pour 200 mL SOB media into each of the shakeflask (one shakeflask per starter culture).	</li>
 <li>	Mark the shakeflasks to match the startercultures	</li>
 <li>	Measure the OD 600 of each starterculture, and inoculate the shakeflask with a volume so the final OD 600 value in the shakeflask culture becomes 0.01	</li>
-<li>	Grow the shakeflask culture at 37 C with shaking. Measure OD values of the sample every 20 minutes once the OD 600 value is above 0.2.	</li>
+<li>	Grow the shakeflask culture at 37 °C with shaking. Measure OD values of the sample every 20 minutes once the OD 600 value is above 0.2.	</li>
-<li>	When OD 600 is between 0.3 and 0.4, put the cultures into an ice bath immediately, and swirl the shakeflash around in the cold water to cool culture. Chill the culture in the icewater for 20-30 minutes, occasionally swirling the cultures.	</li>
+<li>	When OD 600 is between 0.3 and 0.4, put the cultures into an ice bath immediately, and swirl the shake flash around in the cold water to cool culture. Chill the culture in the ice water for 20-30 minutes, occasionally swirling the cultures.	</li>
 <li>	FROM THIS STAGE ON, KEEP CELLS AT ICE/4 °C AT ALL TIMES	</li>
 <li>	For each shakeflask culture, pour the culture into 3 x 50 mL frosted falcon tubes from the freezer.	</li>
 <li>	Keep the tubes on ice	</li>
-<li>	Centrifuge falcon tubes at 3000 x g for 15 min at 4 C (Spin #1 of 4)	</li>
+<li>	Centrifuge falcon tubes at 3000 x g for 15 min at 4 °C (Spin #1 of 4)	</li>
-<li>	Discard supernatant, and resuspend cells in 15 mL icecold 0.1 M MgCl2	</li>
+<li>	Discard supernatant, and resuspend cells in 15 mL icecold 0.1 M MgCl<sub>2</sub>	</li>
 <li>	Keep tubes with cells on ice	</li>
 <li>	Pool the resuspended cells into one of their matching falcon tubes, so you now have 3 different 50 mL falcon tubes, one with cells corresponding to each of the starter cultures you had.	</li>
 <li>	Keep tubes on ice	</li>
-<li>	Centrifuge falcon tubes at 2000 x g for 15 min at 4 o C (Spin #2 of 4)	</li>
+<li>	Centrifuge falcon tubes at 2000 x g for 15 min at 4 °C (Spin #2 of 4)	</li>
-<li>	Discard the supernatant, and resuspend pellet in 40 mL icecold 0.1 M CaCl2	</li>
+<li>	Discard the supernatant, and resuspend pellet in 40 mL icecold 0.1 M CaCl<sub>2</sub>	</li>
 <li>	Keep tubes on ice	</li>
 <li>	Let cell suspensions stand in ice for 20-30 minutes	</li>
-<li>	Centrifuge falcon tubes at 2000 x g for 15 min at 4 C (Spin #3 of 4)	</li>
+<li>	Centrifuge falcon tubes at 2000 x g for 15 min at 4 °C (Spin #3 of 4)	</li>
-<li>	Discard supernatant, and resuspend pellet in 10 mL icecold 85 mM CaCl 2 + 15 % glycerol	</li>
+<li>	Discard supernatant, and resuspend pellet in 10 mL icecold 85 mM CaCl<sub>2</sub> + 15 % glycerol	</li>
 <li>	Keep tubes on ice	</li>
-<li>	Centrifuge falcon tubes at 1000 x g for 15 minutes at 4 C (Spin #4 of 4)	</li>
+<li>	Centrifuge falcon tubes at 1000 x g for 15 minutes at 4 °C (Spin #4 of 4)	</li>
 <li>	Pellet might look small and will be a bit fragile. Handle tubes with care when taking them out of centrifuge	</li>
 <li>	Attention: The next few steps are best done on ice inside a LAF bench	</li>
-<li>	Resuspend pellet in 800 uL ice-cold 85 mM CaCl2 + 15 % glycerol	</li>
+<li>	Resuspend pellet in 800 µL ice-cold 85 mM CaCl<sub>2</sub> + 15 % glycerol	</li>
 <li>	Put falcon tubes on ice	</li>
-<li>	Immediately after cells are confirmed resuspended, aliquot 30 uL of the competent cell culture into the chilled PCR tubes	</li>
+<li>	Immediately after cells are confirmed resuspended, aliquot 30 µL of the competent cell culture into the chilled PCR tubes	</li>
 <li>	Put tubes into -80 °C freezer as fast as possible	</li>
@@ Line 692: / Line 937: @@
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/c/ce/T--DTU-Denmark--E_coli_compentent_cells.pdf" download="Competent-cells-protocol.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
@@ Line 701: / Line 945: @@
+<!--#############################################################################################
+Fluorescence Measurements
+##############################################################################################-->
+<button id="FluorescenceMeasurements" class="collapsible">Fluorescence measurements</button>
+<div class="excontent">
+<div class="row propadding">
+<div class="col-xs-12">
+<p>
+Here, a description of the instruments, settings, and general protocol for the fluorescence measurements can be found.
+</p>
+</div>
+<div class="col-sm-4 col-xs-12">
+<h3 class="media heading">Materials</h3>
+<h4> Materials </h4>
+<ul class="protocolli">
+	<li> SpectraMax iD3 or equivalent plate reader</li>
+</ul>
+<h4> Consumables </h4>
+<ul class="protocolli">
+	<li> Corning® Clear Polystyrene 96-Well Microplate </li>
+</ul>
+</div>
+<div class="col-sm-8 col-xs-12 protoco1">
+<h3 class="media heading">Procedure</h3>
+<h4> Fluorescence measurements </h4>
+<ul>
+	<li> If not done already, 100 µL of sample was loaded into the well of a spectophotometry plate. </li>
+	<li> The RFP was measured using the following settings: (Ex/Em) </li>
+		<ul>
+			<li>mCherry: 580/625</li>
+			<li>GFP: 488/530</li>
+		</ul>
+	</ul>
+</div>
+<h3> Click below for a lab friendly version of this protocol</h3>
+<a href="https://static.igem.org/mediawiki/2019/7/73/T--DTU-Denmark--Fluorescens_measurements.pdf" download="Fluorescence-measurements.pdf">
+    <button class="butn protocolbtn">Download File</button>
+</a>
+</div>
+</div>
@@ Line 707: / Line 1,003: @@
 Gibson Assembly Protocol (combined)
 ##############################################################################################-->
-<button id="GibsonAssemblyProtocol" class="collapsible">Gibson Assembly(combined)</button>
+<button id="GibsonAssemblyProtocol" class="collapsible">Gibson assembly (combined)</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 718: / Line 1,014: @@
 Protocol for MiniPrep adapted from Qiagen's <a href="https://www.qiagen.com/us/resources/download.aspx?id=56b0162c-23b0-473c-9229-12e8b5c8d590&lang=en" target="_blank">QIAprep® Spin Miniprep Kit</a>
 </p>
 </div>
@@ Line 854: / Line 1,148: @@
    <tr>
      <th class="tg-udlr">Component</th>
-     <th class="tg-udlr">50 µl Reaction</th>
+     <th class="tg-udlr">50 µL Reaction</th>
    </tr>
    <tr>
      <td class="tg-0pky">Component</td>
-     <td class="tg-0pky">50 µl Reaction</td>
+     <td class="tg-0pky">50 µL Reaction</td>
    </tr>
    <tr>
@@ Line 866: / Line 1,160: @@
    <tr>
      <td class="tg-0pky">10X CutSmart Buffer</td>
-     <td class="tg-0pky">5 µl (1X)</td>
+     <td class="tg-0pky">5 µL (1X)</td>
    </tr>
    <tr>
      <td class="tg-y698">DpnI</td>
-     <td class="tg-y698">1.0 µl (or 10 units)</td>
+     <td class="tg-y698">1.0 µL (or 10 units)</td>
    </tr>
    <tr>
      <td class="tg-0pky">Nuclease-free Water</td>
-     <td class="tg-0pky">to 50 µl</td>
+     <td class="tg-0pky">to 50 µL</td>
    </tr>
 </table>
 <ul  start="4" class="protocolli">
-	<li>Incubate at 37°C for 5–15 minutes (DpnI is Time-Saver qualified.)</li>
+	<li>Incubate at 37 °C for 5–15 minutes (DpnI is Time-Saver qualified).</li>
 		<li>Optional: Heat inactivate at 80 °C for 20 min if not doing a DNA purification step</li>
 </ul>
@@ Line 886: / Line 1,180: @@
 <h4>PCR Purification</h4>
 <ul start="6" class="protocolli">
-	<li>	Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10 µl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow	</li>
+	<li>	Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10 µL 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow	</li>
-<li>	Place a QIAquick column in a provided 2 ml collection tube	</li>
+<li>	Place a QIAquick column in a provided 2 mL collection tube	</li>
 <li>	To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s. Discard flow-through and place the QIAquick column back in the same tube.	</li>
-<li>	To wash, add 750 µl Buffer PE to the QIAquick column centrifuge for 30–60 s. Discard flow-through and place the QIAquick column back into the same tube.	</li>
+<li>	To wash, add 750 µL Buffer PE to the QIAquick column centrifuge for 30–60 s. Discard flow-through and place the QIAquick column back into the same tube.	</li>
-<li>	Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 1 min to remove residual wash buffer.	</li>
+<li>	Centrifuge the QIAquick column once more in the provided 2 mL collection tube for 1 min to remove residual wash buffer.	</li>
-<li>	Place each QIAquick column in a clean 1.5 ml microcentrifuge tube.	</li>
+<li>	Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.	</li>
-<li>	To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min and then centrifuge.	</li>
+<li>	To elute DNA, add 50 µL Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µL elution buffer to the center of the QIAquick membrane, let the column stand for 1 min and then centrifuge.	</li>
 </ul>
@@ Line 913: / Line 1,207: @@
 <li>On ice, combine<br>5 µL DNA and 5 µL Master Mix for the 2X kit or<br>7.5 µL DNA and 2.5 µL Master Mix for the HC 4X kit<br>Mix well andbriefly centrifuge</li>
 <li>Incubate at 50 °C for 1 hour</li>
-<li>Store reactions at -20 °C or use in 1-2 uL per reaction for transformation</li>
+<li>Store reactions at -20 °C or use in 1-2 µL per reaction for transformation</li>
+</ul>
+<h4>Transformation protocol</h4>
+<ul start="3" class="protocolli">
+	<li>	Thaw chemically-competent cells on ice	</li>
+<li>	Add 2 µL of the chilled assembly product to the competent cells. Mix gently by pipetting up or down or by flicking the tube 4-5 times. Do NOT vortex	</li>
+<li>	Place the mixture on ice for 30 minutes. Do not mix	</li>
+<li>	Heat shock at 42 °C for 30 seconds. Do not mix	</li>
+<li>	Transfer tubes to ice for 2 minutes	</li>
+<li>	Add 950 µL of room temperature SOC media to the tubes	</li>
+<li>	Incubate the tube for 37 °C for 60 minutes. shake vigerously (250 rpm) or rotate	</li>
+<li>	Warm selection plates to 37 °C	</li>
+<li>	Spread 100 µL of the cells onto the selection plates.	</li>
+<li>	Note: Use Amp plates for the positive control	</li>
+<li>	Incubate overnight at 37 °C.	</li>
 </ul>
+<h4>Day 2:</h4>
+<ul start="3" class="protocolli">
+	<li>Plate counting: Count CFU and control plates</li>
+        <li>Innoculate LB + antibiotic media for O/N culture</li>
+</ul>
+<h4>Day 3: Miniprep</h4>
+<ul start="3" class="protocolli">
+	<li>Use a miniprep kit to purify the assembled plasmids.</li>
+</ul>
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/6/6e/T--DTU-Denmark--Gibson_Assembly.pdf" download="Gibson-Assembly-full-procedure.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
@@ Line 939: / Line 1,265: @@
 Golden gate assembly with BsaI (Type IIs assembly)
 ##############################################################################################-->
-<button id="ggBsaI" class="collapsible">Golden gate assembly with BsaI (Type IIs assembly)</button>
+<button id="ggBsaI" class="collapsible">Golden Gate assembly with BsaI (Type IIs assembly)</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 945: / Line 1,271: @@
 <p>
-This protocol is made to assembly parts with TypeIIs enzyme BsaI HF v2. These parts will most probably have the standard Moclo overhangs. Made by PHS, adapted from <a href="https://static.igem.org/mediawiki/2017/e/e8/T--Evry_Paris-Saclay--protocol--pdf--gate.pdf" target="_blank">here</a>.
+This protocol is made to assembly parts with TypeIIs enzyme BsaI HF v2. These parts will have the standard MoClo overhangs. Protocol adapted by Philip Sørensen from a <a href="https://static.igem.org/mediawiki/2017/e/e8/T--Evry_Paris-Saclay--protocol--pdf--gate.pdf" target="_blank">Evry Paris-Saclay's protocol</a>.
 </p>
@@ Line 959: / Line 1,285: @@
 <li>	10X T4 DNA ligase buffer	</li>
 <li>	BsaI HF v2 enzyme (NEB)	</li>
-<li>	Reciever plasmid (BsaI compatible like pGIA2P2o )	</li>
+<li>	Reciever plasmid</li>
 <li>	DNA fragments with BsaI overhangs	</li>
-<li>	MilliQ water	</li>
+<li>	Milli-Q water	</li>
 </ul>
-<h3 class="media heading">Media</h3>
@@ Line 971: / Line 1,295: @@
 <ul class="protocolli">
 	<li>	PCR tubes	</li>
-<li>	10uL pipette tips	</li>
+<li>	10 µL pipette tips	</li>
 <li>	Thermocycler	</li>
 </ul>
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
@@ Line 993: / Line 1,317: @@
 - Equimolar amounts of inserts		<br>
-- MilliQ for a total volume of 20 µL.		<br></li>
+- Milli-Q for a total volume of 20 µL.		<br></li>
 	<li>Mix gently</li>
 <li>Place the tube on a thermocycler</li>
@@ Line 1,021: / Line 1,345: @@
    </tr>
    <tr>
-     <td class="tg-0pky">1: Activation of BsaI HF v2</td>
+     <td class="tg-0pky">Activation of BsaI HF v2</td>
      <td class="tg-vqzh">37 °C </td>
      <td class="tg-73oq">5 min</td>
    </tr>
    <tr>
-     <td class="tg-0pky">2: Activation of T4 ligase</td>
+     <td class="tg-0pky">Activation of T4 ligase</td>
      <td class="tg-vqzh">16 °C </td>
      <td class="tg-73oq">5 min</td>
@@ Line 1,036: / Line 1,360: @@
    </tr>
    <tr>
-     <td class="tg-0pky">3: Inactivation BsaI HF v2</td>
+     <td class="tg-0pky">Inactivation BsaI HF v2</td>
      <td class="tg-vqzh">65 °C </td>
      <td class="tg-73oq">20 min</td>
    </tr>
    <tr>
-     <td class="tg-y698">4: Inactivation T4 ligase</td>
+     <td class="tg-y698">Inactivation T4 ligase</td>
      <td class="tg-rbwf">85 °C </td>
      <td class="tg-vfn0">10 min</td>
    </tr>
    <tr>
-     <td class="tg-0pky">5: Hold </td>
+     <td class="tg-0pky">Hold </td>
      <td class="tg-vqzh">4 °C </td>
      <td class="tg-73oq">Hold</td>
    </tr>
 </table>
+<ul>
+   <li>The cloned plasmids can then be transformed as described in the <a href="#HifiDNAass">Hifi assembly and transformation protocol.</a></li>
+</ul>
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/f/fa/T--DTU-Denmark--Golden_Gate_BsaI.pdf" download="Golden-Gate-BsaI.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
 </div>
@@ Line 1,075: / Line 1,393: @@
 Golden gate assembly with SapI (Type IIs assembly)
 ##############################################################################################-->
-<button id="ggaSapI" class="collapsible">Golden gate assembly with SapI (Type IIs assembly)</button>
+<button id="ggaSapI" class="collapsible">Golden Gate assembly with SapI (Type IIs assembly)</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 1,081: / Line 1,399: @@
 <p>
-This protocol is made to assembly parts with TypeIIs enzyme SapI
+This protocol is made to assembly parts with TypeIIs enzyme SapI. Protocol adapted by Philip Sørensen from a <a href="https://static.igem.org/mediawiki/2017/e/e8/T--Evry_Paris-Saclay--protocol--pdf--gate.pdf" target="_blank">Evry Paris-Saclay's protocol</a>.
 </p>
 </div>
@@ Line 1,095: / Line 1,411: @@
 <li>	10X T4 DNA ligase buffer	</li>
 <li>	SapI enzyme (NEB)	</li>
-<li>	Reciever plasmid (Sap one compatible like Inaccessible DNA Sequence )	</li>
+<li>	Reciever plasmid</li>
 <li>	DNA fragments with SapI overhangs	</li>
-<li>	MilliQ water	</li>
+<li>	Milli-Q water	</li>
 </ul>
@@ Line 1,104: / Line 1,420: @@
 <ul class="protocolli">
 	<li>	PCR tubes	</li>
-<li>	10uL pipette tips	</li>
+<li>	10 µL pipette tips	</li>
 <li>	Thermocycler	</li>
@@ Line 1,110: / Line 1,426: @@
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
@@ Line 1,127: / Line 1,443: @@
 - Equimolar amounts of inserts	<br>
-- MilliQ for a total volume of 20 µL.	<br></li>
+- Milli-Q for a total volume of 20 µL.	<br></li>
 	<li>Mix gently</li>
 <li>Place the tube on a thermocycler</li>
@@ Line 1,154: / Line 1,470: @@
    </tr>
    <tr>
-     <td class="tg-0pky">1: Activation of SapI</td>
+     <td class="tg-0pky">Activation of SapI</td>
-     <td class="tg-vqzh">37&nbsp;&nbsp;°C </td>
+     <td class="tg-vqzh">37 °C </td>
      <td class="tg-73oq">5 min</td>
    </tr>
    <tr>
-     <td class="tg-0pky">2: Activation of T4 ligase</td>
+     <td class="tg-0pky">Activation of T4 ligase</td>
-     <td class="tg-vqzh">16&nbsp;&nbsp;°C </td>
+     <td class="tg-vqzh">16 °C </td>
      <td class="tg-73oq">5 min</td>
    </tr>
@@ Line 1,169: / Line 1,485: @@
    </tr>
    <tr>
-     <td class="tg-0pky">3: Inactivation SapI</td>
+     <td class="tg-0pky">Inactivation SapI</td>
      <td class="tg-vqzh">65&nbsp;&nbsp;°C </td>
      <td class="tg-73oq">20 min</td>
    </tr>
    <tr>
-     <td class="tg-y698">4: Inactivation T4 ligase</td>
+     <td class="tg-y698">Inactivation T4 ligase</td>
      <td class="tg-rbwf">85&nbsp;&nbsp;°C </td>
      <td class="tg-vfn0">10 min</td>
    </tr>
    <tr>
-     <td class="tg-0pky">5: Hold </td>
+     <td class="tg-0pky">Hold </td>
      <td class="tg-vqzh">4&nbsp;&nbsp;°C </td>
      <td class="tg-73oq">Hold</td>
    </tr>
 </table>
+<ul>
+   <li>The cloned plasmids can then be transformed as described in the <a href="#HifiDNAass">Hifi assembly and transformation protocol.</a></li>
+</ul>
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/c/c4/T--DTU-Denmark--Golden_Gate_SapI.pdf" download="Golden-Gate-SapI.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
 </div>
@@ Line 1,201: / Line 1,519: @@
 Hifi DNA assembly and Transformation
 ##############################################################################################-->
-<button id="HifiDNAass" class="collapsible">Hifi DNA assembly and Transformation</button>
+<button id="HifiDNAass" class="collapsible">Hifi DNA assembly and transformation</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 1,207: / Line 1,525: @@
 <p>
-Adapted by Jacob Mejlsted & Joen Haahr Jensen from IDT's <a href="https://international.neb.com/-/media/catalog/datacards-or-manuals/manuale2621.pdf" target="_blank">HiFi assembly protocol</a>.
+Adapted by Jacob Mejlsted & Joen Haahr Jensen from NEB's <a href="https://international.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol" target="_blank">HiFi assembly protocol</a>.
 </p>
@@ Line 1,232: / Line 1,547: @@
 <ul class="protocolli">
 	<li>	Hifi DNA assembly Master mix	</li>
-<li>	Steril MilliQ water	</li>
+<li>	Sterile Milli-Q water	</li>
-<li>	Competent E. coli	</li>
+<li>	Competent <i>E. coli</i> cells	</li>
-<li>	Prepared DNA fragments for assembly (See information on primer construction)	</li>
+<li>	Prepared DNA fragments for assembly</li>
 </ul>
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
 <h4>Assembly protocol</h4>
 <ul class="protocolli">
-	<li>Set up the following reaction on ice:<br>* If the inserts are less less than 200 bp, use a 5 fold excess of inserts instead of a 2 fold excess	<br>
+	<li>Set up the following reaction on ice:<br>* If the inserts are less than 200 bp, use a 5 fold excess of inserts instead of a 2 fold excess	<br>
-** If a greater number of fragments are assembled, increase the volumen of the reaction and use additional HiFi DNA assembly master mix	<br></li>
+** If a greater number of fragments are assembled, increase the volume of the reaction and use additional HiFi DNA assembly master mix	<br></li>
 </ul>
@@ Line 1,267: / Line 1,582: @@
      <td class="tg-kftd"></td>
      <td class="tg-1wig">2-3 Fragments*</td>
-     <td class="tg-1wig">4-6 Fragements</td>
+     <td class="tg-1wig">4-6 Fragements**</td>
      <td class="tg-1wig">Postive control</td>
    </tr>
@@ Line 1,278: / Line 1,593: @@
    <tr>
      <td class="tg-kftd">Total amount of DNA fragments</td>
-     <td class="tg-0lax">0.03-0.3pmols<br>X uL</td>
+     <td class="tg-0lax">0.03-0.3pmols<br>X µL</td>
-     <td class="tg-0lax">0.2-0.5 pmols<br>X uL</td>
+     <td class="tg-0lax">0.2-0.5 pmols<br>X µL</td>
-     <td class="tg-0lax">10 uL</td>
+     <td class="tg-0lax">10 µL</td>
    </tr>
    <tr>
      <td class="tg-kftd">NEB Hifi Assembly master mix</td>
-     <td class="tg-kftd">10 uL</td>
+     <td class="tg-kftd">10 µL</td>
-     <td class="tg-kftd">10 uL</td>
+     <td class="tg-kftd">10 µL</td>
-     <td class="tg-kftd">10 uL</td>
+     <td class="tg-kftd">10 µL</td>
    </tr>
    <tr>
-     <td class="tg-kftd">MilliQ water</td>
+     <td class="tg-kftd">Milli-Q water</td>
-     <td class="tg-0lax">10-X uL</td>
+     <td class="tg-0lax">10-X µL</td>
-     <td class="tg-0lax">10-x uL</td>
+     <td class="tg-0lax">10-x µL</td>
-     <td class="tg-0lax">0 uL</td>
+     <td class="tg-0lax">0 µL</td>
    </tr>
    <tr>
      <td class="tg-kftd">Total volume</td>
-     <td class="tg-kftd">20 uL</td>
+     <td class="tg-kftd">20 µL</td>
-     <td class="tg-kftd">20 uL</td>
+     <td class="tg-kftd">20 µL</td>
-     <td class="tg-kftd">20 uL</td>
+     <td class="tg-kftd">20 µL</td>
    </tr>
 </table>
@@ Line 1,305: / Line 1,620: @@
 <ul  start="2" class="protocolli">
-	<li>Incubate the reaction samples in a thermocycler at 50oC for 15 minutes (when 2-3 fragments are assembled) or 60 minutes (when 4-6 fragments are assembled). Following incubation, store the reaction samples at -20oC for subsequent transformation<br>Note: Extended incubation up to 60 minutes can in some cases improve transformation efficiency</li>
+	<li>Incubate the reaction samples in a thermocycler at 50 °C for 15 minutes (when 2-3 fragments are assembled) or 60 minutes (when 4-6 fragments are assembled). Following incubation, store the reaction samples at -20 °C for subsequent transformation<br>Note: Extended incubation up to 60 minutes can in some cases improve transformation efficiency</li>
 </ul>
@@ Line 1,314: / Line 1,629: @@
 	<li>	Thaw chemically-competent cells on ice	</li>
-<li>	Add 2 uL of the chilled assembly product to the competent cells. Mix gently by pipetting up or down or by flicking the tube 4-5 times. Do NOT vortex	</li>
+<li>	Add 2 µL of the chilled assembly product to the competent cells. Mix gently by pipetting up or down or by flicking the tube 4-5 times. Do NOT vortex	</li>
 <li>	Place the mixture on ice for 30 minutes. Do not mix	</li>
-<li>	Heat shock at 42oC for 30 seconds. Do not mix	</li>
+<li>	Heat shock at 42 °C for 30 seconds. Do not mix	</li>
 <li>	Transfer tubes to ice for 2 minutes	</li>
-<li>	Add 950 uL of room temperature SOC media to the tubes	</li>
+<li>	Add 950 µL of room temperature SOC media to the tubes	</li>
-<li>	Incubate the tube for 37oC for 60 minutes. shake vigerously (250 rpm) or rotate	</li>
+<li>	Incubate the tube for 37 °C for 60 minutes. shake vigerously (250 rpm) or rotate	</li>
-<li>	Warm selection plates to 37oC	</li>
+<li>	Warm selection plates to 37 °C	</li>
-<li>	Spread 100uL of the cells onto the selection plates.	</li>
+<li>	Spread 100µL of the cells onto the selection plates.	</li>
 <li>	Note: Use Amp plates for the positive control	</li>
-<li>	Incubate overnight at 37oC.	</li>
+<li>	Incubate overnight at 37 °C.	</li>
 </ul>
@@ Line 1,339: / Line 1,654: @@
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/7/7f/T--DTU-Denmark--Hifi_DNA_assembly.pdf" download="Hifi-assembly.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
@@ Line 1,347: / Line 1,661: @@
 </div>
+<!--#############################################################################################
+Minimal media
+##############################################################################################-->
+<button id="Minimal-media" class="collapsible">Minimal media</button>
+<div class="excontent">
+<div class="row propadding">
+<div class="col-xs-12">
+<p>
+Protocol for production of 1 liter of minimal media
+</p>
+</div>
+<div class="col-sm-4 col-xs-12">
+<h3 class="media heading">Materials</h3>
+<h4>Materials </h4>
+<ul class="protocolli">
+	<li>50 mL of 20% w/V D-glucose stock</li>
+<li>50 mL of 20x nitrate salts stock</li>
+<li>1 mL 1000x Trace elements stock</li>
+<li>1 mL of 1% thiamine stock</li>
+<li>20 g agar</li>
+</ul>
+</div>
+<div class="col-sm-8 col-xs-12 protoco1">
+<h3 class="media heading">Procedure</h3>
+<h4>Media preparation</h4>
+<ul>
+	<li>Mix ingredients in flask and add MiliQ water to 1 litre</li>
+	<li>Mix with magnet stirrer until the sugar is dissolved</li>
+	<li>Autoclave at 121 °C for 20 min</li>
+</ul>
+</div>
+<h3> Click below for a lab friendly version of this protocol</h3>
+<a href="https://static.igem.org/mediawiki/2019/f/f0/T--DTU-Denmark--Minimal_media.pdf" download="Minimal-media.pdf">
+    <button class="butn protocolbtn">Download File</button>
+</a>
+</div>
+</div>
+<!--#############################################################################################
+Miscellaneous protocols
+##############################################################################################-->
+<button id="Miscellaneousprotocols" class="collapsible">Miscellaneous protocols</button>
+<div class="excontent">
+<div class="row propadding">
+<div class="col-xs-12">
+<p>
+Here is a list of miscellaneous protocols, all of which were performed according to the manufacturer's and/or the referenced specifications. <a href="" target="_blank"></a>.
+</p>
+</div>
+<div class="col-sm-8 col-xs-12 protoco1">
+<h3 class="media heading">Protocols</h3>
+<h4> Gel electrophoresis </h4>
+<ul>
+	<li>Gels contained 1% agarose and were stained with 1000X SYBR Safe (Invitrogen) </li>
+	<li>DNA fragment solutions were combined with 6X Purple Loading Dye (New England Biolabs). </li>
+<li> Samples were run in 1X TAE running buffer at 100 V with a 1kb DNA ladder (New England Biolabs). </li>
+<li> Electrophoresis was run on the RunOne Electrophoresis System (Embi Tec). </li>
+<li> Pictures of the resulting gels were taken with the ChemiDoc™ XRS+ Imaging System or equivalent.
+ </li>
+</ul>
+<h4> Gel purifications </h4>
+<ul start="3">
+	<li> Isolated DNA bands were purified via the QIAquick Gel Extraction kit (QIAGEN). </li>
+</ul>
+</ul>
+<h4> PCR spin purifications </h4>
+<ul start="3">
+	<li>When deemed necessary, plasmid DNA was spin purified, rather than gel purified, via the QIAquick PCR Purification Kit (QIAGEN). </li>
+</ul>
+</ul>
+<h4> Plasmid miniprep purification </h4>
+<ul start="3">
+	<li> Plasmids were purified using the QIAprep Spin Miniprep Kit (QIAGEN). </li>
+</ul>
+</ul>
+<h4> DNA sequencing </h4>
+<ul start="3">
+	<li> DNA was verified by Sanger sequencing, using the Eurofins’ overnight sanger sequencing service. </li>
+</ul>
+</ul>
+<h4> Primer/DNA resuspension </h4>
+<ul start="3">
+	<li> Primers were diluted to a stock concentration of 100 µM and a working concentration of 10 µM. </li>
+	<li> Synthetic DNA (e.g. synthetic promoters) were diluted to 25 ng/µL or 40 ng/µL, depending on downstream uses.</li>
+</ul>
+</div>
+</div>
+</div>
@@ Line 1,360: / Line 1,776: @@
 <p>
-This protocol is for linearising backbones with the PacI+Nt.BbvCI USER casette. This will allow assembly of PCR product with USER tails to be assembled into this linearised plasmid
+This protocol is for linearising backbones with the PacI+Nt.BbvCI USER casette. This will allow assembly of PCR product with USER tails to be assembled into this linearised plasmid.
 </p>
@@ Line 1,371: / Line 1,787: @@
 <ul class="protocolli">
-	<li>	Plasmid: 40 uL	</li>
+	<li>	Plasmid: 40 µL	</li>
-<li>	PacI: 2 uL	</li>
+<li>	PacI: 2 µL	</li>
-<li>	Cutsmart buffer (NEB): 10 uL	</li>
+<li>	Cutsmart buffer (NEB): 10 µL	</li>
-<li>	MQ water: 48 uL	</li>
+<li>	MQ water: 48 µL	</li>
 </ul>
@@ Line 1,381: / Line 1,797: @@
 <ul class="protocolli">
 	<li>	Finished digest I	</li>
-<li>	Nt.BbvCI: 2uL	</li>
+<li>	Nt.BbvCI: 2 µL	</li>
 </ul>
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
@@ Line 1,396: / Line 1,812: @@
 <h4>Digest II</h4>
 <ul  start="3" class="protocolli">
-	<li>Take the digest I reaction from the 37 °Cincubator, add in the 2 uL Nt.BbvCI to the reaction mixture, and incubate for 2 hours at 37 °C</li>
+	<li>Take the digest I reaction from the 37 °C incubator, add in the 2 µL Nt.BbvCI to the reaction mixture, and incubate for 2 hours at 37 °C</li>
 	<li>Heat inactivate the samples at 80 °C for 30 min</li>
@@ Line 1,403: / Line 1,819: @@
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/7/7a/T--DTU-Denmark--USER_digestion.pdf" download="USER-linearization.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
 </div>
@@ Line 1,419: / Line 1,830: @@
 PCR Protocol for Phusion® High-Fidelity DNA Polymerase
 ##############################################################################################-->
-<button id="Phusion1" class="collapsible">PCR Protocol for Phusion® High-Fidelity DNA Polymerase</button>
+<button id="Phusion1" class="collapsible">PCR protocol for Phusion® high-fidelity DNA polymerase</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 1,453: / Line 1,864: @@
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
@@ Line 1,472: / Line 1,883: @@
    <tr>
      <th class="tg-il2e">Component</th>
-     <th class="tg-il2e">20 µl Reaction</th>
+     <th class="tg-il2e">20 µL Reaction</th>
-     <th class="tg-il2e">50 µl Reaction</th>
+     <th class="tg-il2e">50 µL Reaction</th>
      <th class="tg-il2e">Final Concentration</th>
    </tr>
    <tr>
      <td class="tg-0lax">Nuclease-free water</td>
-     <td class="tg-0lax">to 20 µl</td>
+     <td class="tg-0lax">to 20 µL</td>
-     <td class="tg-0lax">to 50 µl</td>
+     <td class="tg-0lax">to 50 µL</td>
      <td class="tg-0lax"></td>
    </tr>
    <tr>
      <td class="tg-kftd">5X Phusion HF or GC Buffer</td>
-     <td class="tg-kftd">4 µl</td>
+     <td class="tg-kftd">4 µL</td>
-     <td class="tg-kftd">10 µl</td>
+     <td class="tg-kftd">10 µL</td>
      <td class="tg-kftd">1X</td>
    </tr>
    <tr>
      <td class="tg-0lax">10 mM dNTPs</td>
-     <td class="tg-0lax">0.4 µl</td>
+     <td class="tg-0lax">0.4 µL</td>
-     <td class="tg-0lax">1 µl</td>
+     <td class="tg-0lax">1 µL</td>
      <td class="tg-0lax">200 µM</td>
    </tr>
    <tr>
      <td class="tg-kftd">10 µM Forward Primer</td>
-     <td class="tg-kftd">1 µl</td>
+     <td class="tg-kftd">1 µL</td>
-     <td class="tg-kftd">2.5 µl</td>
+     <td class="tg-kftd">2.5 µL</td>
      <td class="tg-kftd">0.5 µM</td>
    </tr>
    <tr>
      <td class="tg-0lax">10 µM Reverse Primer</td>
-     <td class="tg-0lax">1 µl</td>
+     <td class="tg-0lax">1 µL</td>
-     <td class="tg-0lax">2.5 µl</td>
+     <td class="tg-0lax">2.5 µL</td>
      <td class="tg-0lax">0.5 µM</td>
    </tr>
@@ Line 1,514: / Line 1,925: @@
    <tr>
      <td class="tg-0lax">DMSO (optional)</td>
-     <td class="tg-0lax">(0.6 µl)</td>
+     <td class="tg-0lax">(0.6 µL)</td>
-     <td class="tg-0lax">(1.5 µl)</td>
+     <td class="tg-0lax">(1.5 µL)</td>
      <td class="tg-0lax">3%</td>
    </tr>
    <tr>
      <td class="tg-kftd">Phusion DNA Polymerase</td>
-     <td class="tg-kftd">0.2 µl</td>
+     <td class="tg-kftd">0.2 µL</td>
-     <td class="tg-kftd">0.5 µl</td>
+     <td class="tg-kftd">0.5 µL</td>
-     <td class="tg-kftd">1.0 units/50 µl PCR</td>
+     <td class="tg-kftd">1.0 units/50 µL PCR</td>
    </tr>
 </table>
@@ Line 1,546: / Line 1,957: @@
    <tr>
      <th class="tg-udlr">Reactant</th>
-     <th class="tg-udlr">Per reaction (50uL) [µl]</th>
+     <th class="tg-udlr">Per reaction (50µL) [µL]</th>
-     <th class="tg-udlr">Mastermix [µl]</th>
+     <th class="tg-udlr">Mastermix [µL]</th>
    </tr>
    <tr>
@@ Line 1,590: / Line 2,001: @@
    </tr>
    <tr>
-     <td class="tg-0lax">MilliQ</td>
+     <td class="tg-0lax">Milli-Q</td>
      <td class="tg-0lax">33.5</td>
      <td class="tg-0lax">335</td>
@@ Line 1,618: / Line 2,029: @@
    <tr>
      <td class="tg-0pky">Initial denaturation</td>
-     <td class="tg-0pky">98 C </td>
+     <td class="tg-0pky">98 °C </td>
      <td class="tg-0pky">30 seconds</td>
      <td class="tg-0pky">1 cycle</td>
@@ Line 1,624: / Line 2,035: @@
    <tr>
      <td class="tg-y698" rowspan="3">Amplification</td>
-     <td class="tg-y698">98 C </td>
+     <td class="tg-y698">98 °C </td>
      <td class="tg-y698">10 seconds</td>
      <td class="tg-y698" rowspan="3">25-30 cycles</td>
@@ Line 1,633: / Line 2,044: @@
    </tr>
    <tr>
-     <td class="tg-y698">72 C</td>
+     <td class="tg-y698">72 °C</td>
      <td class="tg-y698">15-30 seconds/kb*</td>
    </tr>
    <tr>
      <td class="tg-0pky">Final extension</td>
-     <td class="tg-0pky">72 C</td>
+     <td class="tg-0pky">72 °C</td>
      <td class="tg-0pky">5-10 minutes</td>
      <td class="tg-0pky">1 cycle</td>
@@ Line 1,644: / Line 2,055: @@
    <tr>
      <td class="tg-y698">Hold</td>
-     <td class="tg-y698">4 C</td>
+     <td class="tg-y698">4 °C</td>
      <td class="tg-y698">-</td>
      <td class="tg-y698">1 cycle</td>
    </tr>
 </table>
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/5/51/T--DTU-Denmark--Phusion_protocol.pdf" download="Phusion-PCR.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
 </div>
@@ Line 1,703: / Line 2,107: @@
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
@@ Line 1,723: / Line 2,127: @@
    <tr>
      <th class="tg-il2e">Component</th>
-     <th class="tg-il2e">25 μl reaction***</th>
+     <th class="tg-il2e">25 μL reaction***</th>
-     <th class="tg-il2e">50 μl reaction***</th>
+     <th class="tg-il2e">50 μL reaction***</th>
      <th class="tg-il2e">Final Concentration</th>
    </tr>
    <tr>
      <td class="tg-0lax">5X OneTaq Standard Reaction Buffer*</td>
-     <td class="tg-0lax">5 µl</td>
+     <td class="tg-0lax">5 µL</td>
-     <td class="tg-0lax">10 μl</td>
+     <td class="tg-0lax">10 μL</td>
      <td class="tg-0lax">1X</td>
    </tr>
    <tr>
      <td class="tg-kftd">10 mM dNTPs (#N0447)</td>
-     <td class="tg-kftd">0.5 µl</td>
+     <td class="tg-kftd">0.5 µL</td>
-     <td class="tg-kftd">1 μl</td>
+     <td class="tg-kftd">1 μL</td>
      <td class="tg-kftd">200 µM</td>
    </tr>
    <tr>
      <td class="tg-0lax">10 µM Forward Primer</td>
-     <td class="tg-0lax">0.5 µl</td>
+     <td class="tg-0lax">0.5 µL</td>
-     <td class="tg-0lax">1 μl</td>
+     <td class="tg-0lax">1 μL</td>
      <td class="tg-0lax">0.2 µM</td>
    </tr>
    <tr>
      <td class="tg-kftd">10 µM Reverse Primer</td>
-     <td class="tg-kftd">0.5 µl</td>
+     <td class="tg-kftd">0.5 µL</td>
-     <td class="tg-kftd">1 μl</td>
+     <td class="tg-kftd">1 μL</td>
      <td class="tg-kftd">0.2 µM</td>
    </tr>
    <tr>
      <td class="tg-0lax">OneTaq DNA Polymerase</td>
-     <td class="tg-0lax">0.125 µl</td>
+     <td class="tg-0lax">0.125 µL</td>
-     <td class="tg-0lax">0.25 µl</td>
+     <td class="tg-0lax">0.25 µL</td>
-     <td class="tg-0lax">1.25 units/50 µl PCR**</td>
+     <td class="tg-0lax">1.25 units/50 µL PCR**</td>
    </tr>
    <tr>
@@ Line 1,765: / Line 2,169: @@
    <tr>
      <td class="tg-0lax">Nuclease-free water</td>
-     <td class="tg-0lax">to 25 µl</td>
+     <td class="tg-0lax">to 25 µL</td>
-     <td class="tg-0lax">to 50 µl</td>
+     <td class="tg-0lax">to 50 µL</td>
      <td class="tg-0lax"></td>
    </tr>
@@ Line 1,773: / Line 2,177: @@
 <p>*OneTaq GC Reaction Buffer and High GC Enhancer can be used for difficult amplicons<br>
-**For plasmids or viral DNA, use 1 pg–10 ng DNA for a 50 µl reaction.
+**For plasmids or viral DNA, use 1 pg–10 ng DNA for a 50 µL reaction.
 <br>
-***It can be advantagous to pool some of the parts into a master mix, as some labs cannot dispence 0.125 µl accurately.
+***It can be advantagous to pool some of the parts into a master mix, as some labs cannot dispense 0.125 µL accurately.
 </p>
@@ Line 1,794: / Line 2,198: @@
    <tr>
      <th class="tg-udlr">Reactant</th>
-     <th class="tg-udlr">Per reaction (50uL) [µl]</th>
+     <th class="tg-udlr">Per reaction (50µL) [µL]</th>
-     <th class="tg-udlr">Mastermix [µl]</th>
+     <th class="tg-udlr">Mastermix [µL]</th>
    </tr>
    <tr>
@@ Line 1,857: / Line 2,261: @@
    <tr>
      <td class="tg-0pky">Initial Denaturation</td>
-     <td class="tg-0pky">94°C</td>
+     <td class="tg-0pky">94 °C</td>
      <td class="tg-0pky">30 seconds</td>
    </tr>
    <tr>
      <td class="tg-y698" rowspan="3">30 Cycles</td>
-     <td class="tg-y698">94°C</td>
+     <td class="tg-y698">94 °C</td>
      <td class="tg-y698">15-30 seconds</td>
    </tr>
    <tr>
-     <td class="tg-0pky">45-68°C</td>
+     <td class="tg-0pky">45-68 °C</td>
      <td class="tg-0pky">15-60 seconds</td>
    </tr>
    <tr>
-     <td class="tg-y698">68°C</td>
+     <td class="tg-y698">68 °C</td>
      <td class="tg-y698">1 minute per kb</td>
    </tr>
    <tr>
      <td class="tg-0pky">Final Extension</td>
-     <td class="tg-0pky">68°C</td>
+     <td class="tg-0pky">68 °C</td>
      <td class="tg-0pky">5 minutes</td>
    </tr>
    <tr>
      <td class="tg-y698">Hold</td>
-     <td class="tg-y698">4-10°C</td>
+     <td class="tg-y698">4-10 °C</td>
      <td class="tg-y698"></td>
    </tr>
@@ Line 1,889: / Line 2,293: @@
 </ul>
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/a/a3/T--DTU-Denmark--OneTaq_protocol.pdf" download="OneTaq-PCR.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
 </div>
@@ Line 1,905: / Line 2,304: @@
 PCR using Q5® High-Fidelity 2X Master Mix
 ##############################################################################################-->
-<button id="PCRQ5hf" class="collapsible">PCR using Q5® High-Fidelity 2X Master Mix</button>
+<button id="PCRQ5hf" class="collapsible">PCR using Q5® high-fidelity 2X master mix</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 1,937: / Line 2,336: @@
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
@@ Line 1,962: / Line 2,361: @@
    <tr>
      <td class="tg-0lax">Q5 High-Fidelity 2X Master Mix</td>
-     <td class="tg-0lax">12.5 µl</td>
+     <td class="tg-0lax">12.5 µL</td>
-     <td class="tg-0lax">25 µl</td>
+     <td class="tg-0lax">25 µL</td>
    </tr>
    <tr>
      <td class="tg-kftd">10 µM Forward Primer</td>
-     <td class="tg-kftd">1.25 µl</td>
+     <td class="tg-kftd">1.25 µL</td>
-     <td class="tg-kftd">2.5 µl</td>
+     <td class="tg-kftd">2.5 µL</td>
    </tr>
    <tr>
      <td class="tg-0lax">10 µM Reverse Primer</td>
-     <td class="tg-0lax">1.25 µl</td>
+     <td class="tg-0lax">1.25 µL</td>
-     <td class="tg-0lax">2.5 µl</td>
+     <td class="tg-0lax">2.5 µL</td>
    </tr>
    <tr>
@@ Line 1,982: / Line 2,381: @@
    <tr>
      <td class="tg-0lax">Nuclease-Free Water</td>
-     <td class="tg-0lax">to 25 µl</td>
+     <td class="tg-0lax">to 25 µL</td>
-     <td class="tg-0lax">to 50 µl</td>
+     <td class="tg-0lax">to 50 µL</td>
    </tr>
    <tr>
@@ Line 2,019: / Line 2,418: @@
    <tr>
      <td class="tg-0lax">Initial denaturation</td>
-     <td class="tg-0lax">98 C</td>
+     <td class="tg-0lax">98 °C</td>
      <td class="tg-0lax">30 seconds</td>
      <td class="tg-0lax">1 cycle</td>
@@ Line 2,025: / Line 2,424: @@
    <tr>
      <td class="tg-kftd" rowspan="3">Amplification</td>
-     <td class="tg-kftd">98 C</td>
+     <td class="tg-kftd">98 °C</td>
      <td class="tg-kftd">10 seconds</td>
      <td class="tg-kftd" rowspan="3">25-30 cycles</td>
@@ Line 2,034: / Line 2,433: @@
    </tr>
    <tr>
-     <td class="tg-kftd">72 C</td>
+     <td class="tg-kftd">72 °C</td>
      <td class="tg-kftd">30 seconds/kb</td>
    </tr>
    <tr>
      <td class="tg-0lax">Final extension</td>
-     <td class="tg-0lax">72 C</td>
+     <td class="tg-0lax">72 °C</td>
      <td class="tg-0lax">2 minutes</td>
      <td class="tg-0lax">1 cycle</td>
@@ Line 2,045: / Line 2,444: @@
    <tr>
      <td class="tg-kftd">Hold</td>
-     <td class="tg-kftd">4 C</td>
+     <td class="tg-kftd">4 °C</td>
      <td class="tg-kftd">-</td>
      <td class="tg-kftd">1 cycle</td>
@@ Line 2,052: / Line 2,451: @@
 <ul start="3" class="protocolli">
-	<li>The PCR products can then be stored at -20 C, used directly, or purified using PCR purification or gel extraction</li>
+	<li>The PCR products can then be stored at -20 °C, used directly, or purified using PCR purification or gel extraction</li>
 </ul>
@@ Line 2,058: / Line 2,457: @@
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/f/f6/T--DTU-Denmark--Q5_master_mix.pdf" download="Q5-master-mix.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
 </div>
 <!--#############################################################################################
-Aspergillus niger protoplast protocol
+PCR using X7 polymerase
 ##############################################################################################-->
-<button id="resuspensionofdna" class="collapsible">Aspergillus niger protoplast</button>
+<button id="PCRusingX7polymerase" class="collapsible">PCR using X7 polymerase</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 2,081: / Line 2,474: @@
 <p>
-This protocol is the adapted protocol from 223.
+This protocol describes the required components and method for a X7 polymerase mediated PCR reaction. <a href="" target="_blank"></a>.
 </p>
 </div>
@@ Line 2,091: / Line 2,482: @@
+<h4> Consumables </h4>
 <ul class="protocolli">
-	<li>	Fungal plates (either streaked or 3-point)	</li>
+	<li> PCR strip/tubes </li>
-<li>	Drigalski spatula	</li>
-<li>	500 ml shake flasks	</li>
-<li>	Counting chamber	</li>
-<li>	Solutions (APB, ATP, PCT, milli-Q, TM)	</li>
-<li>	30 °C incubator with shaking	</li>
-<li>	sterile tea spoon	</li>
-<li>	mira cloth in funnel (sterile)	</li>
-<li>	Glucanex	</li>
-<li>	magnet stirrer	</li>
-<li>	magnets	</li>
-<li>	50 ml sterile falcon tubes	</li>
-<li>	0.45 µm filters	</li>
-<li>	50 ml syringe	</li>
-<li>	centrifuge for falcon tubes	</li>
-</ul>
-<h3 class="media heading">Media</h3>
-<ul class="protocolli">
-	<li>	Aspergillus transformation buffer (ATB)	</li>
-<li>	Aspergillus protoplastation buffer (APB)	</li>
-<li>	500 ml shake flasks	</li>
-<li>	YPD media	</li>
 </ul>
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
-<h4>Initiation</h4>
+<h4>PCR Amplfication of DNA Fragments</h4>
 <ul class="protocolli">
-	<li>Streak spore suspension of host strain on YPD plates supplemented with uridine (for this particular fungus) and let grow for a week (there should be black spores!)</li>
+	<li>Prepare PCR reaction (see table below)</li>
-	<li>All solutions should be sterile.</li>
 </ul>
-<h4>Day 1 (inoculation)</h4>
-<ul  start="3" class="protocolli">
+<style type="text/css">
-	<li>Add 95 ml of YPD media supplemented with uridine to a shake flask and transfer 5 ml of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 ml. It is a good idea to make more than one shake flask at a time.</li>
+.tg  {border-collapse:collapse;border-spacing:0;border:none;}
-	<li>Incubate shake flasks at 30 °C, 150 RMP for 48 h.</li>
+.tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:0px;overflow:hidden;word-break:normal;}
+.tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:0px;overflow:hidden;word-break:normal;}
+.tg .tg-il2e{font-weight:bold;background-color:#09314f;color:#ffffff;text-align:left;vertical-align:top}
+.tg .tg-kftd{background-color:#efefef;text-align:left;vertical-align:top}
+.tg .tg-0lax{text-align:left;vertical-align:top}
+</style>
+<table class="tg">
+  <tr>
+    <th class="tg-il2e">COMPONENT</th>
+    <th class="tg-il2e">50 µL REACTION</th>
+  </tr>
+  <tr>
+    <td class="tg-0lax">CXL buffer</td>
+    <td class="tg-0lax">10 µL</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">10 mM dNTPs</td>
+    <td class="tg-kftd">1 µL</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax">10 µM Forward Primer</td>
+    <td class="tg-0lax">2.5 µL</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">10 µM Reverse Primer</td>
+    <td class="tg-kftd">2.5 µL</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax">Template DNA</td>
+    <td class="tg-0lax">Variable (often 1 µL)</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">Pfu X7 Polymerase</td>
+    <td class="tg-kftd">0.5 µL</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax">DMSO</td>
+    <td class="tg-0lax">1.5 µL</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">Nuclease-Free Water</td>
+    <td class="tg-kftd">to 50 µL</td>
+  </tr>
+</table>
 </ul>
-<h4>Day 3 (mycelial harvest)</h4>
-<ul start="5" class="protocolli">
+<ul  start="2" class="protocolli">
-	<li>Place the sterile funnel with a mira cloth in to a sterile blue cap bottle and transfer the contents of the shake flasks to the mira cloth (content should be brown and thick).</li>
+	<li>Run reaction in a thermocycler</li>
-	<li>Wash the mycelia using Aspergillus protoplastation buffer (APB) to remove residual glucose from the mycelia (this can inhibit protoplastation). You need to use quite a bit of APB. squeeze out remaining liquid using a sterile spoon. Then, transfer the mycelium to falcon tubes ( ≈2 g per tube => 1 shake flask ≈ 2 falcon tubes). </li>
 </ul>
-<h4> Protoplastation </h4>
+<h4> Thermocycler PCR program </h4>
-<ul  start="7" class="protocolli">
+<ul>
-	<li>Add glucanex to APB to get a final concentration of 40 mg glucanex per ml APB and dissolve glucanex via gentle magnetic stirring and no heat.</li>
-	<li>Sterile filter 20 ml of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 ml syringe (there is a bit of resistance in the filter but that's ok).</li>
+<style type="text/css">
-	<li>Shake/incubate enzyme-mycelium mix at 30 °C, 150 RPM for 2-3 h.</li>
+.tg  {border-collapse:collapse;border-spacing:0;border:none;}
-<li>From now on, whenever you pipette anything with the cells in it, cut of the edge of the pipette tip and CAREFULLY(!) pipette the cells. If you don't do this, they break as they don't have a cell wall to keep them stable.</li>
+.tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:0px;overflow:hidden;word-break:normal;}
-<li>Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/ml). Approved protoplast solutions are then diluted by pouring APB up to 40 ml. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile milli-Q H2O and carefully place 5 ml of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)</li>
+.tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:0px;overflow:hidden;word-break:normal;}
-<li>In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 ml and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.</li>
+.tg .tg-il2e{font-weight:bold;background-color:#09314f;color:#ffffff;text-align:left;vertical-align:top}
-<li>Count protoplasts in microscope by diluting a small sample 1:100.</li>
+.tg .tg-kftd{background-color:#efefef;text-align:left;vertical-align:top}
-<li>Resuspend protoplasts in 4 ml ATB to obtain concentrated solution.</li>
+.tg .tg-0lax{text-align:left;vertical-align:top}
+</style>
+<table class="tg">
+  <tr>
+    <th class="tg-il2e">Step</th>
+    <th class="tg-il2e">Temperature</th>
+    <th class="tg-il2e">Duration</th>
+    <th class="tg-il2e">Number of Cycles</th>
+  </tr>
+  <tr>
+    <td class="tg-kftd">Initial denaturation</td>
+    <td class="tg-kftd">95 °C</td>
+    <td class="tg-kftd">2 minutes</td>
+    <td class="tg-kftd">1 cycle</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax" rowspan="3">Amplification</td>
+    <td class="tg-0lax">95 °C</td>
+    <td class="tg-0lax">30 seconds</td>
+    <td class="tg-0lax" rowspan="3">25-30 cycles</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">Primer Tm</td>
+    <td class="tg-kftd">30 seconds</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax">72 °C</td>
+    <td class="tg-0lax">around 1 min/kb*</td>
+  </tr>
+  <tr>
+    <td class="tg-kftd">Final extension</td>
+    <td class="tg-kftd">72 °C</td>
+    <td class="tg-kftd">5 minutes</td>
+    <td class="tg-kftd">1 cycle</td>
+  </tr>
+  <tr>
+    <td class="tg-0lax">Hold</td>
+    <td class="tg-0lax">4 °C</td>
+    <td class="tg-0lax">-</td>
+    <td class="tg-0lax">1 cycle</td>
+  </tr>
+</table>
+<li>The PCR products can then be stored at -20 °C, used directly, or purified using PCR purification or gel extraction.</li>
 </ul>
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/6/66/T--DTU-Denmark--X7_polymerase_protocol.pdf" download="X7-polymerase-protocol.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
 </div>
 <!--#############################################################################################
-Aspergillus niger protoplast protocol
+Protein extraction protocol
 ##############################################################################################-->
-<button id="resuspensionofdna" class="collapsible">Aspergillus niger protoplast</button>
+<button id="Protein-extraction" class="collapsible">Protein extraction</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 2,180: / Line 2,635: @@
 <p>
-This protocol is the adapted protocol from 223.
+This protocol is an adaptation of a GUS (β-glucuranidase) assay provided by Zofia Dorota Jarczynska and adapted by Jacob Mejlsted.
 </p>
 </div>
@@ Line 2,189: / Line 2,642: @@
 <h3 class="media heading">Materials</h3>
+<h4>Extraction buffer (1L) </h4>
 <ul class="protocolli">
-	<li>	Fungal plates (either streaked or 3-point)	</li>
+	<li>50 mM Na<sub>3</sub>PO<sub>4</sub>, pH 7 (50 mL of 1 M stock solution)</li>
-<li>	Drigalski spatula	</li>
+	<li>10 mM β-mercaptoethanol (0.7 mL of 14.4 M stock solution)</li>
-<li>	500 ml shake flasks	</li>
+	<li>10 mM Na<sub>2</sub>EDTA (20 mL of 0.5 M Na<sub>2</sub>EDTA stock solution)</li>
-<li>	Counting chamber	</li>
+	<li>0.1% Sodium Lauryl Saccosine (10 mL of 10% Sarcosyl)</li>
-<li>	Solutions (APB, ATP, PCT, milli-Q, TM)	</li>
+	<li>0.1% Triton X-100 (10 mL f 10% Triton)</li>
-<li>	30 °C incubator with shaking	</li>
+	<li>Milli-Q water up to 1L</li>
-<li>	sterile tea spoon	</li>
-<li>	mira cloth in funnel (sterile)	</li>
-<li>	Glucanex	</li>
-<li>	magnet stirrer	</li>
-<li>	magnets	</li>
-<li>	50 ml sterile falcon tubes	</li>
-<li>	0.45 µm filters	</li>
-<li>	50 ml syringe	</li>
-<li>	centrifuge for falcon tubes	</li>
 </ul>
-<h3 class="media heading">Media</h3>
+<h4>Equipment</h4>
+<ul class="protocolli">
+	<li>Centrifuge with cooling</li>
+	<li>Plate reader</li>
+	<li>Homogenizer</li>
+	<li>Beads (big, metal)</li>
+	<li>Sterile funnels with mira cloth</li>
+</ul>
+<h4>Chemicals</h4>
 <ul class="protocolli">
-	<li>	Aspergillus transformation buffer (ATB)	</li>
+	<li>Bradford reagent (Like SigmaAldrich B6916)</li>
-<li>	Aspergillus protoplastation buffer (APB)	</li>
+	<li>BSA standard (Bovine Serum Albumin 2 mg/mL)</li>
-<li>	500 ml shake flasks	</li>
-<li>	YPD media	</li>
 </ul>
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
-<h4>Initiation</h4>
+<h4>Protein Extraction</h4>
-<ul class="protocolli">
+<ul>
-	<li>Streak spore suspension of host strain on YPD plates supplemented with uridine (for this particular fungus) and let grow for a week (there should be black spores!)</li>
+	<li><b>CRITICAL</b>          Set the centrifuge at 4 °C</li>
-	<li>All solutions should be sterile.</li>
+	<li>Seperate biomass from supernantant using mira cloth or other methods</li>
+	<li>Take ~100 mg biomass and place in a FastPrep tube with one big metal bead</li>
-</ul>
+	<li>Place the tubes in liquid nitrogen</li>
+	<li>Homogenize for 1 min at 45 Hz</li>
+	<li>Add 500 µL extraction buffer</li>
+	<li>Homogenize for 2 min at 45 Hz</li>
+	<li>Centrifuge for 2 min at 10,000 x g at 4 °C</li>
+	<li>Collect the liquid phase and place in a new tube</li>
+	<li>Centrifuge for 15 min at 10,000 x g at 4 °C</li>
+	<li>Aliquote the supernantant into new tubes</li>
+<li>Store at -80 °C</li>
-<h4>Day 1 (inoculation)</h4>
-<ul  start="3" class="protocolli">
-	<li>Add 95 ml of YPD media supplemented with uridine to a shake flask and transfer 5 ml of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 ml. It is a good idea to make more than one shake flask at a time.</li>
-	<li>Incubate shake flasks at 30 °C, 150 RMP for 48 h.</li>
 </ul>
+<h4>Bradford Assay</h4>
+<ul start="3">
-<h4>Day 3 (mycelial harvest)</h4>
+	<li>Transfer 100 µL protein sample into a microtiter place</li>
-<ul start="5" class="protocolli">
+	<li>Measure fluorescense at the appripriate wavelenghts</li>
-	<li>Place the sterile funnel with a mira cloth in to a sterile blue cap bottle and transfer the contents of the shake flasks to the mira cloth (content should be brown and thick).</li>
-	<li>Wash the mycelia using Aspergillus protoplastation buffer (APB) to remove residual glucose from the mycelia (this can inhibit protoplastation). You need to use quite a bit of APB. squeeze out remaining liquid using a sterile spoon. Then, transfer the mycelium to falcon tubes ( ≈2 g per tube => 1 shake flask ≈ 2 falcon tubes). </li>
 </ul>
+<h4>Fluorescence Assay (optional)</h4>
-<h4> Protoplastation </h4>
+<ul start="3">
-<ul  start="7" class="protocolli">
+	<li>Mix 20 µL protein sample with 230 µL Bradfor reagent</li>
-	<li>Add glucanex to APB to get a final concentration of 40 mg glucanex per ml APB and dissolve glucanex via gentle magnetic stirring and no heat.</li>
+	<li>Transfer to a new microtiter plate</li>
-	<li>Sterile filter 20 ml of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 ml syringe (there is a bit of resistance in the filter but that's ok).</li>
+	<li>Measure the blue color at 595 nm</li>
-	<li>Shake/incubate enzyme-mycelium mix at 30 °C, 150 RPM for 2-3 h.</li>
+	<li><b>Remember to include a standard curve and Bradford reagent</b></li>
-<li>From now on, whenever you pipette anything with the cells in it, cut of the edge of the pipette tip and CAREFULLY(!) pipette the cells. If you don't do this, they break as they don't have a cell wall to keep them stable.</li>
+	<li>Determine protein concentration</li>
-<li>Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/ml). Approved protoplast solutions are then diluted by pouring APB up to 40 ml. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile milli-Q H2O and carefully place 5 ml of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)</li>
-<li>In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 ml and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.</li>
-<li>Count protoplasts in microscope by diluting a small sample 1:100.</li>
-<li>Resuspend protoplasts in 4 ml ATB to obtain concentrated solution.</li>
 </ul>
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/a/a9/T--DTU-Denmark--Protein_extraction_protocol.pdf" download="Protein-extraction-protocol.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
 </div>
 <!--#############################################################################################
-Aspergillus niger protoplast protocol
+Retrieving DNA from the iGEM distribution kit
 ##############################################################################################-->
-<button id="resuspensionofdna" class="collapsible">Aspergillus niger protoplast</button>
+<button id="RetrievingDNAfrom" class="collapsible">Retrieving DNA from the iGEM distribution kit</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 2,278: / Line 2,723: @@
 <p>
-This protocol is the adapted protocol from 223.
+Adapted by Jacob Mejlsted from <a href="http://parts.igem.org/Help:Protocols/Transformation" target="_blank">iGEM's protocol</a>
 </p>
@@ Line 2,286: / Line 2,731: @@
 <div class="col-sm-4 col-xs-12">
 <h3 class="media heading">Materials</h3>
+<h4>Materials</h4>
 <ul class="protocolli">
-	<li>	Fungal plates (either streaked or 3-point)	</li>
+	<li>	Resuspended DNA to be transformed	</li>
-<li>	Drigalski spatula	</li>
+<li>	10 pg/µL Positive transformation control DNA (e.g. pSB1C3 w/ BBa_J04450, RFP on high-copy chloramphenicol resistant plasmid. Located in the Competent Cell Test Kit.)	</li>
-<li>	500 ml shake flasks	</li>
+<li>	Competent Cells (50 µL per sample)	</li>
-<li>	Counting chamber	</li>
+<li>	1.5 mL Microtubes	</li>
-<li>	Solutions (APB, ATP, PCT, milli-Q, TM)	</li>
+<li>	SOC Media (950 µL per sample)	</li>
-<li>	30 °C incubator with shaking	</li>
+<li>	Petri plates w/ LB agar and antibiotic (2 per sample)	</li>
-<li>	sterile tea spoon	</li>
-<li>	mira cloth in funnel (sterile)	</li>
-<li>	Glucanex	</li>
-<li>	magnet stirrer	</li>
-<li>	magnets	</li>
-<li>	50 ml sterile falcon tubes	</li>
-<li>	0.45 µm filters	</li>
-<li>	50 ml syringe	</li>
-<li>	centrifuge for falcon tubes	</li>
 </ul>
-<h3 class="media heading">Media</h3>
+<h4>Equipment</h4>
 <ul class="protocolli">
-	<li>	Aspergillus transformation buffer (ATB)	</li>
+	<li>	Floating Foam Tube Rack	</li>
-<li>	Aspergillus protoplastation buffer (APB)	</li>
+<li>	Ice & ice bucket	</li>
-<li>	500 ml shake flasks	</li>
+<li>	Lab Timer	</li>
-<li>	YPD media	</li>
+<li>	42 °C water bath	</li>
+<li>	37 °C incubator	</li>
+<li>	Sterile spreader or glass beads	</li>
+<li>	Microcentrifuge	</li>
 </ul>
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
-<h4>Initiation</h4>
+<h4>Method: Day 1</h4>
 <ul class="protocolli">
-	<li>Streak spore suspension of host strain on YPD plates supplemented with uridine (for this particular fungus) and let grow for a week (there should be black spores!)</li>
+	<li>	Resuspend DNA in selected wells in the Distribution Kit with 10 µL dH<sub>2</sub>O. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.	</li>
-	<li>All solutions should be sterile.</li>
+<li>	Label 1.5 mL tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5 mL tubes (one tube for each transformation, including your control) in a floating foam tube rack.	</li>
-</ul>
+<li>	Thaw competent cells on ice: This may take 10-15 min for a 260 µL stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.	</li>
-<h4>Day 1 (inoculation)</h4>
+<li>	Pipette 50 µL of competent cells into 1.5 mL tube: 50 µL in a 1.5 mL tube per transformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 1.5 mL tube for your control.	</li>
-<ul  start="3" class="protocolli">
-	<li>Add 95 ml of YPD media supplemented with uridine to a shake flask and transfer 5 ml of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 ml. It is a good idea to make more than one shake flask at a time.</li>
+<li>	Pipette 1 µL of resuspended DNA into 1.5 mL tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.	</li>
-	<li>Incubate shake flasks at 30 °C, 150 RMP for 48 h.</li>
+<li>	Pipette 1 µL of control DNA into 2 mL tube: Pipette 1 µL of 10 pg/µL control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.	</li>
+<li>	Close 1.5 mL tubes, incubate on ice for 30 min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.</li>
+<li>	Heat shock tubes at 42 °C for 45 sec: 1.5 mL tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.	</li>
+<li>	Incubate on ice for 5 min: Return transformation tubes to ice bucket.	</li>
+<li>	Pipette 950 µL SOC media to each transformation: SOC should be stored at 4 °C, but can be warmed to room temperature before use. Check for contamination.	</li>
+<li>	Incubate at 37 °C for 1 hours, shaking at 200-300 rpm	</li>
+<li>	Pipette 100 µL of each transformation onto petri plates. Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.	</li>
+<li>	Incubate transformations overnight (14-18 hours) at 37 °C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.	</li>
 </ul>
+<h4>Method: Day 2</h4>
-<h4>Day 3 (mycelial harvest)</h4>
+<ul  start="14" class="protocolli">
-<ul start="5" class="protocolli">
+	<li>	Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make <a href="#proto3aassembly">glycerol stocks</a>, grow up cell cultures and miniprep.	</li>
-	<li>Place the sterile funnel with a mira cloth in to a sterile blue cap bottle and transfer the contents of the shake flasks to the mira cloth (content should be brown and thick).</li>
-	<li>Wash the mycelia using Aspergillus protoplastation buffer (APB) to remove residual glucose from the mycelia (this can inhibit protoplastation). You need to use quite a bit of APB. squeeze out remaining liquid using a sterile spoon. Then, transfer the mycelium to falcon tubes ( ≈2 g per tube => 1 shake flask ≈ 2 falcon tubes). </li>
+<li>	Count colonies for control transformation: Count colonies on the 100 μL control plate and <a href="http://parts.igem.org/Help:Competent_Cell_Test_Kit" target="_blank">calculate your competent cell efficiency</a>. Competent cells should have an efficiency of 1.5x10<sup>8</sup> to 6x10<sup>8</sup> CFU/µg DNA.	</li>
 </ul>
-<h4> Protoplastation </h4>
-<ul  start="7" class="protocolli">
-	<li>Add glucanex to APB to get a final concentration of 40 mg glucanex per ml APB and dissolve glucanex via gentle magnetic stirring and no heat.</li>
-	<li>Sterile filter 20 ml of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 ml syringe (there is a bit of resistance in the filter but that's ok).</li>
-	<li>Shake/incubate enzyme-mycelium mix at 30 °C, 150 RPM for 2-3 h.</li>
-<li>From now on, whenever you pipette anything with the cells in it, cut of the edge of the pipette tip and CAREFULLY(!) pipette the cells. If you don't do this, they break as they don't have a cell wall to keep them stable.</li>
-<li>Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/ml). Approved protoplast solutions are then diluted by pouring APB up to 40 ml. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile milli-Q H2O and carefully place 5 ml of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)</li>
-<li>In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 ml and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.</li>
-<li>Count protoplasts in microscope by diluting a small sample 1:100.</li>
-<li>Resuspend protoplasts in 4 ml ATB to obtain concentrated solution.</li>
-</ul>
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/b/bd/T--DTU-Denmark--Distribution_kit_DNA.pdf" download="Distribution-kit-extraction.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
 </div>
 <!--#############################################################################################
-Aspergillus niger protoplast protocol
+Sampling procedures
 ##############################################################################################-->
-<button id="resuspensionofdna" class="collapsible">Aspergillus niger protoplast</button>
+<button id="Samplingprocedures" class="collapsible">Sampling procedures</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 2,376: / Line 2,816: @@
 <p>
-This protocol is the adapted protocol from 223.
+Here, the procedure for sampling of the shake flask and bioreactor cultivations is described.
 </p>
 </div>
@@ Line 2,385: / Line 2,823: @@
 <h3 class="media heading">Materials</h3>
+<h4> Consumables </h4>
 <ul class="protocolli">
-	<li>	Fungal plates (either streaked or 3-point)	</li>
+	<li> 10 mL syringe (bioreactor) </li>
-<li>	Drigalski spatula	</li>
+<li> 15 mL falcon tube </li>
-<li>	500 ml shake flasks	</li>
+<li> 1.5 mL eppendorf tube </li>
-<li>	Counting chamber	</li>
+	<li> Corning® Clear Polystyrene 96-Well Microplate </li>
-<li>	Solutions (APB, ATP, PCT, milli-Q, TM)	</li>
-<li>	30 °C incubator with shaking	</li>
-<li>	sterile tea spoon	</li>
-<li>	mira cloth in funnel (sterile)	</li>
-<li>	Glucanex	</li>
-<li>	magnet stirrer	</li>
-<li>	magnets	</li>
-<li>	50 ml sterile falcon tubes	</li>
-<li>	0.45 µm filters	</li>
-<li>	50 ml syringe	</li>
-<li>	centrifuge for falcon tubes	</li>
 </ul>
-<h3 class="media heading">Media</h3>
-<ul class="protocolli">
-	<li>	Aspergillus transformation buffer (ATB)	</li>
-<li>	Aspergillus protoplastation buffer (APB)	</li>
-<li>	500 ml shake flasks	</li>
-<li>	YPD media	</li>
-</ul>
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
-<h4>Initiation</h4>
+<h4> Shake flask cultivation </h4>
-<ul class="protocolli">
+<ul>
-	<li>Streak spore suspension of host strain on YPD plates supplemented with uridine (for this particular fungus) and let grow for a week (there should be black spores!)</li>
+	<li> In a clean bench, approximately 1 mL of sample containing biomass was sampled via pipetting. </li>
-	<li>All solutions should be sterile.</li>
+	<li> 100 µL sample containing biomass was transferred to a spectrophotometry plate.  </li>
+	<li> 200 µL of sample was spun down at 10.000 g for 3 min and 100 µL transferred to a spectrophotometry plate. </li>
+	<li> Immediately after sampling, samples in the spectrophotometry plate was measured for fluorescence according to the <a href="#FluorescenceMeasurements">fluorescence measurement protocol</a>, and the remainder of the sample in the eppendorf tubes stored at -20 °C.   </li>
 </ul>
-<h4>Day 1 (inoculation)</h4>
-<ul  start="3" class="protocolli">
-	<li>Add 95 ml of YPD media supplemented with uridine to a shake flask and transfer 5 ml of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 ml. It is a good idea to make more than one shake flask at a time.</li>
-	<li>Incubate shake flasks at 30 °C, 150 RMP for 48 h.</li>
-</ul>
-<h4>Day 3 (mycelial harvest)</h4>
+<h4>Bioreactor cultivation </h4>
-<ul start="5" class="protocolli">
+<ul start="3">
-	<li>Place the sterile funnel with a mira cloth in to a sterile blue cap bottle and transfer the contents of the shake flasks to the mira cloth (content should be brown and thick).</li>
+	<li> For sampling of the bioreactor, extra care was taken to prevent contamination. </li>
-	<li>Wash the mycelia using Aspergillus protoplastation buffer (APB) to remove residual glucose from the mycelia (this can inhibit protoplastation). You need to use quite a bit of APB. squeeze out remaining liquid using a sterile spoon. Then, transfer the mycelium to falcon tubes ( ≈2 g per tube => 1 shake flask ≈ 2 falcon tubes). </li>
+	<li> The sterile 10 mL syringe was sprayed with 70% ethanol and inserted in the sampling tube. </li>
+<li> The clamps on the sampling tube were loosened and 5 mL sample was drawn and discarded.  </li>
+<li> The syringe was again sprayed with 70% ethanol, and reinserted in the sampling tube. </li>
+	<li> Approximately 10 mL sample containing biomass was drawn and transferred to a 15 mL falcon tube. </li>
+<li> The clamps on the sampling tube were tightened, and the sampling tube sprayed with 70% ethanol.  </li>
+	<li> The samples were stored at -20 C for later protein purification. </li>
 </ul>
-<h4> Protoplastation </h4>
-<ul  start="7" class="protocolli">
-	<li>Add glucanex to APB to get a final concentration of 40 mg glucanex per ml APB and dissolve glucanex via gentle magnetic stirring and no heat.</li>
-	<li>Sterile filter 20 ml of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 ml syringe (there is a bit of resistance in the filter but that's ok).</li>
-	<li>Shake/incubate enzyme-mycelium mix at 30 °C, 150 RPM for 2-3 h.</li>
-<li>From now on, whenever you pipette anything with the cells in it, cut of the edge of the pipette tip and CAREFULLY(!) pipette the cells. If you don't do this, they break as they don't have a cell wall to keep them stable.</li>
-<li>Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/ml). Approved protoplast solutions are then diluted by pouring APB up to 40 ml. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile milli-Q H2O and carefully place 5 ml of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)</li>
-<li>In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 ml and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.</li>
-<li>Count protoplasts in microscope by diluting a small sample 1:100.</li>
-<li>Resuspend protoplasts in 4 ml ATB to obtain concentrated solution.</li>
-</ul>
 </div>
 <!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/3/39/T--DTU-Denmark--Sampling_procedures.pdf" download="Sampling-procedures.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
 </div>
@@ Line 2,466: / Line 2,873: @@
 <!--#############################################################################################
-Retreving DNA from the iGEM distribution kit
+Shake flask cultivation
 ##############################################################################################-->
-<button id="RetrevingDNAfrom" class="collapsible">Retreving DNA from the iGEM distribution kit</button>
+<button id="Shakeflaskcultivation" class="collapsible">Shake flask cultivation</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 2,474: / Line 2,881: @@
 <p>
-Adapted by Jacob Mejlsted from <a href="http://parts.igem.org/Help:Protocols/Transformation" target="_blank">iGEM's protocol</a>
+Here, we describe the protocol for the set-up and execution of the shake flask cultivations. Please, <a href="#Minimal-media" >click here</a> for the media recipe, <a href="#Samplingprocedures">here</a> for sampling procedures for the specifics regarding sampling, and <a href="#FluorescenceMeasurements">here</a> for details regarding the fluorescence measurements.
 </p>
 </div>
 <div class="col-sm-4 col-xs-12">
 <h3 class="media heading">Materials</h3>
-<h4>Materials</h4>
+<h4> Materials </h4>
 <ul class="protocolli">
-	<li>	Resuspended DNA to be transformed	</li>
+	<li> Pyrex shake flasks (with baffles) </li>
-<li>	10pg/µl Positive transformation control DNA (e.g. pSB1C3 w/ BBa_J04450, RFP on high-copy chloramphenicol resistant plasmid. Located in the Competent Cell Test Kit.)	</li>
+	<li> Minimal media </li>
-<li>	Competent Cells (50µl per sample)	</li>
+<li> Spore suspension </li>
-<li>	1.5mL Microtubes	</li>
-<li>	SOC Media (950µL per sample)	</li>
-<li>	Petri plates w/ LB agar and antibiotic (2 per sample)	</li>
 </ul>
-<h4>Equipment</h4>
+<h4> Consumables </h4>
 <ul class="protocolli">
-	<li>	Floating Foam Tube Rack	</li>
+	<li> Cotton stopper </li>
-<li>	Ice & ice bucket	</li>
+<li> Tin foil </li>
-<li>	Lab Timer	</li>
-<li>	42°C water bath	</li>
-<li>	37°C incubator	</li>
-<li>	Sterile spreader or glass beads	</li>
-<li>	Microcentrifuge	</li>
 </ul>
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
-<h4>Method: Day 1</h4>
+<h4> Preparations and inoculation </h4>
-<ul class="protocolli">
+<ul>
-	<li>	Resuspend DNA in selected wells in the Distribution Kit with 10µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.	</li>
+	<li> A breathable cotton ball was added to the mouth of a glass shake flask (with baffles), covered with tin foil, and then autoclaved. </li>
+	<li> 150 mL minimal media was added to each shake flask. </li>
-<li>	Label 1.5ml tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5ml tubes (one tube for each transformation, including your control) in a floating foam tube rack.	</li>
+	<li> The shake flasks were inoculated with their respective spore solutions to a final concentration of 10<sup>6</sup> spores/mL. </li>
+	<li> Shake flasks were incubated for 5 days as described below. </li>
-<li>	Thaw competent cells on ice: This may take 10-15min for a 260µl stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.	</li>
-<li>	Pipette 50µl of competent cells into 1.5ml tube: 50µl in a 1.5ml tube per transformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 1.5ml tube for your control.	</li>
-<li>	Pipette 1µl of resuspended DNA into 1.5ml tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.	</li>
-<li>	Pipette 1µl of control DNA into 2ml tube: Pipette 1µl of 10pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.	</li>
-<li>	Close 1.5ml tubes, incubate on ice for 30min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.	</li>
-<li>	Heat shock tubes at 42°C for 45 sec: 1.5ml tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.	</li>
-<li>	Incubate on ice for 5min: Return transformation tubes to ice bucket.	</li>
-<li>	Pipette 950µl SOC media to each transformation: SOC should be stored at 4°C, but can be warmed to room temperature before use. Check for contamination.	</li>
-<li>	Incubate at 37°C for 1 hours, shaking at 200-300rpm	</li>
-<li>	Pipette 100µL of each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.	</li>
-<li>	Incubate transformations overnight (14-18hr) at 37°C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.	</li>
-</ul>
-<h4>Method: Day 2</h4>
+</ul>
-<ul  start="14" class="protocolli">
+<h4> Conditions </h4>
-	<li>	Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make <a href="https://benchling.com/cwor/f/WDq916Ia-protocols/prt-4NdFZx5B-bacterial-glycerol-stock/edit" target="_blank">glycerol stocks</a>, grow up cell cultures and <a href="https://benchling.com/cwor/f/WDq916Ia-protocols/prt-tav3Oqna-qiaprep-spin-miniprep-kit/edit" target="_blank">miniprep</a>.	</li>
+<ul start="3">
+	<li> Shake flasks were incubated at 30 °C at 150 rpm for 5 days. </li>
-<li>	Count colonies for control transformation: Count colonies on the 100μl control plate and <a href="http://parts.igem.org/Help:Competent_Cell_Test_Kit" target="_blank">calculate your competent cell efficiency</a>. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.	</li>
+	<li> Samples were taken as regularly as described in sampling procedures. </li>
 </ul>
+</div>
+<h3> Click below for a lab friendly version of this protocol</h3>
+<a href="https://static.igem.org/mediawiki/2019/2/25/T--DTU-Denmark--Shake_flask_cultivation.pdf" download="Shake-flask-cultivation.pdf">
+    <button class="butn protocolbtn">Download File</button>
+</a>
 </div>
 </div>
-</div>
 <!--#############################################################################################
-Aspergillus niger protoplast protocol
+Spore suspensions
 ##############################################################################################-->
-<button id="resuspensionofdna" class="collapsible">Aspergillus niger protoplast</button>
+<button id="Sporesuspensions" class="collapsible">Spore suspensions</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 2,566: / Line 2,942: @@
 <p>
-This protocol is the adapted protocol from 223.
+The following is a quick and easy protocol for creating the spore suspensions used for storage and the inoculation of the shake flask, biolector, and bioreactor cultivations. <a href="" target="_blank"></a>.
 </p>
 </div>
@@ Line 2,575: / Line 2,949: @@
 <h3 class="media heading">Materials</h3>
+<h4> Materials </h4>
 <ul class="protocolli">
-	<li>	Fungal plates (either streaked or 3-point)	</li>
+        <li>Fully grown fungi plates (with spores)</li>
-<li>	Drigalski spatula	</li>
+        <li>Drigalski spatula</li>
-<li>	500 ml shake flasks	</li>
+        <li>96% ethanol</li>
-<li>	Counting chamber	</li>
+        <li>FireBoy or other flame-sterilizing equipment</li>
-<li>	Solutions (APB, ATP, PCT, milli-Q, TM)	</li>
+        <li>Sterile Milli-Q water</li>
-<li>	30 °C incubator with shaking	</li>
+        <li>Sterile mira cloth</li>
-<li>	sterile tea spoon	</li>
+        <li>Sterile falcon tubes</li>
-<li>	mira cloth in funnel (sterile)	</li>
+        <li>P1000 pipette and tips</li>
-<li>	Glucanex	</li>
+	<li>Scissors</li>
-<li>	magnet stirrer	</li>
+	<li>Microscope</li>
-<li>	magnets	</li>
+	<li>Counting chamber</li>
-<li>	50 ml sterile falcon tubes	</li>
-<li>	0.45 µm filters	</li>
-<li>	50 ml syringe	</li>
-<li>	centrifuge for falcon tubes	</li>
-</ul>
-<h3 class="media heading">Media</h3>
-<ul class="protocolli">
-	<li>	Aspergillus transformation buffer (ATB)	</li>
-<li>	Aspergillus protoplastation buffer (APB)	</li>
-<li>	500 ml shake flasks	</li>
-<li>	YPD media	</li>
 </ul>
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
-<h4>Initiation</h4>
+<h4> Creating the spore suspensions </h4>
-<ul class="protocolli">
+<ul>
-	<li>Streak spore suspension of host strain on YPD plates supplemented with uridine (for this particular fungus) and let grow for a week (there should be black spores!)</li>
+	<li>Add mira cloth to a falcon tube.</li>
-	<li>All solutions should be sterile.</li>
+	<li>Pour 2-3 mL of Milli-Q water onto a plate</li>
+	<li>Clean and sterilize a drigalski spatula using ethanol and fire.</li>
-</ul>
+	<li>Let the drigalski cool off in the water before commencing the scraping. Then, carefully scrape off the spores from the plate. This should be done with the lid still on and VERY careully as the spores are hydrophobic and would much rather just fly around than being suspended into the water.</li>
+	<li>When all of the spores have been suspended into the water, transfer the liquid to the mira cloth using a pipette with the tip cut off.</li>
+	<li>When this is done, count the spores in the microscope. Usually, a 1000x dilution is necessary.</li>
+	<li> The spore suspension can be kept for up to 3 months at 4 °C. </li>
-<h4>Day 1 (inoculation)</h4>
-<ul  start="3" class="protocolli">
-	<li>Add 95 ml of YPD media supplemented with uridine to a shake flask and transfer 5 ml of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 ml. It is a good idea to make more than one shake flask at a time.</li>
-	<li>Incubate shake flasks at 30 °C, 150 RMP for 48 h.</li>
 </ul>
-<h4>Day 3 (mycelial harvest)</h4>
-<ul start="5" class="protocolli">
-	<li>Place the sterile funnel with a mira cloth in to a sterile blue cap bottle and transfer the contents of the shake flasks to the mira cloth (content should be brown and thick).</li>
-	<li>Wash the mycelia using Aspergillus protoplastation buffer (APB) to remove residual glucose from the mycelia (this can inhibit protoplastation). You need to use quite a bit of APB. squeeze out remaining liquid using a sterile spoon. Then, transfer the mycelium to falcon tubes ( ≈2 g per tube => 1 shake flask ≈ 2 falcon tubes). </li>
-</ul>
-<h4> Protoplastation </h4>
-<ul  start="7" class="protocolli">
-	<li>Add glucanex to APB to get a final concentration of 40 mg glucanex per ml APB and dissolve glucanex via gentle magnetic stirring and no heat.</li>
-	<li>Sterile filter 20 ml of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 ml syringe (there is a bit of resistance in the filter but that's ok).</li>
-	<li>Shake/incubate enzyme-mycelium mix at 30 °C, 150 RPM for 2-3 h.</li>
-<li>From now on, whenever you pipette anything with the cells in it, cut of the edge of the pipette tip and CAREFULLY(!) pipette the cells. If you don't do this, they break as they don't have a cell wall to keep them stable.</li>
-<li>Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/ml). Approved protoplast solutions are then diluted by pouring APB up to 40 ml. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile milli-Q H2O and carefully place 5 ml of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)</li>
-<li>In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 ml and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.</li>
-<li>Count protoplasts in microscope by diluting a small sample 1:100.</li>
-<li>Resuspend protoplasts in 4 ml ATB to obtain concentrated solution.</li>
-</ul>
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/8/85/T--DTU-Denmark--Spore_suspensions.pdf" download="Spore-suspension.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
 </div>
@@ Line 2,667: / Line 3,001: @@
 Protocol for production of 1 liter of fungal transformation media
 </p>
 </div>
@@ Line 2,676: / Line 3,008: @@
 <ul class="protocolli">
-	<li>	342,30 g Sucrose	</li>
+	<li>	342.30 g sucrose	</li>
-<li>	50.0 ml Nitrate salts	</li>
+<li>	50.0 mL Nitrate salts	</li>
-<li>	1 ml Trace metal elements	</li>
+<li>	1 mL Trace metal elements	</li>
-<li>	1 ml Thiamine	</li>
+<li>	1 mL Thiamine	</li>
 <li>	21 g Bacto agar	</li>
 </ul>
@@ Line 2,686: / Line 3,018: @@
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
 <ul class="protocolli">
-	<li>	Mix ingridients in flask and add MiliQ water to 1 litre.	</li>
+	<li>	Mix ingredients in flask and add MiliQ water to 1 litre.	</li>
 <li>	Mix with magnet stirrer until the sugar is dissolved.	</li>
@@ Line 2,701: / Line 3,033: @@
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/8/8e/T--DTU-Denmark--Transformation_media.pdf" download="Transformation-media.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
 </div>
 <!--#############################################################################################
-Aspergillus niger protoplast protocol
+USER-cloning
 ##############################################################################################-->
-<button id="resuspensionofdna" class="collapsible">Aspergillus niger protoplast</button>
+<button id="USER-cloning" class="collapsible">USER-cloning</button>
 <div class="excontent">
 <div class="row propadding">
@@ Line 2,722: / Line 3,051: @@
 <p>
-This protocol is the adapted protocol from 223.
+The following describes the USER-cloning protocol used during our project.
 </p>
 </div>
@@ Line 2,731: / Line 3,058: @@
 <h3 class="media heading">Materials</h3>
+<h4> Reagents </h4>
 <ul class="protocolli">
-	<li>	Fungal plates (either streaked or 3-point)	</li>
+	<li> USER-linearized insert(s) </li>
-<li>	Drigalski spatula	</li>
+	<li> USER-linearized backbone </li>
-<li>	500 ml shake flasks	</li>
+	<li> USER enzyme </li>
-<li>	Counting chamber	</li>
+<li> CutSmart buffer </li>
-<li>	Solutions (APB, ATP, PCT, milli-Q, TM)	</li>
+<li> Milli-Q water </li>
-<li>	30 °C incubator with shaking	</li>
-<li>	sterile tea spoon	</li>
-<li>	mira cloth in funnel (sterile)	</li>
-<li>	Glucanex	</li>
-<li>	magnet stirrer	</li>
-<li>	magnets	</li>
-<li>	50 ml sterile falcon tubes	</li>
-<li>	0.45 µm filters	</li>
-<li>	50 ml syringe	</li>
-<li>	centrifuge for falcon tubes	</li>
 </ul>
-<h3 class="media heading">Media</h3>
+<h4> Consumables </h4>
 <ul class="protocolli">
-	<li>	Aspergillus transformation buffer (ATB)	</li>
+	<li> PCR-strip/tubes </li>
-<li>	Aspergillus protoplastation buffer (APB)	</li>
-<li>	500 ml shake flasks	</li>
-<li>	YPD media	</li>
 </ul>
 </div>
-<div class="col-sm-8 col-xs-12">
+<div class="col-sm-8 col-xs-12 protoco1">
 <h3 class="media heading">Procedure</h3>
-<h4>Initiation</h4>
+<h4> Preparing USER-reaction </h4>
-<ul class="protocolli">
+<ul>
-	<li>Streak spore suspension of host strain on YPD plates supplemented with uridine (for this particular fungus) and let grow for a week (there should be black spores!)</li>
+	<li> In a PCR-tube, combine;
-	<li>All solutions should be sterile.</li>
+	<ul>
+		<li>200 ng USER-linearized insert(s)</li>
+<li>100 ng USER-linearized vector backbone</li>
+<li>1 µL 10X CutSmart buffer</li>
+<li>1 µL USER enzyme</li>
+<li>H2O to 10 µL. </li>
 </ul>
+	<li> Incubate reaction mix as described below. </li>
-<h4>Day 1 (inoculation)</h4>
-<ul  start="3" class="protocolli">
+</ul>
-	<li>Add 95 ml of YPD media supplemented with uridine to a shake flask and transfer 5 ml of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 ml. It is a good idea to make more than one shake flask at a time.</li>
+<h4> Incubating reaction mix </h4>
-	<li>Incubate shake flasks at 30 °C, 150 RMP for 48 h.</li>
+<ul start="3">
+	<li> In a PCR-machine, incubate as follows. </li>
+	<ul>
+<li>37 °C for 25 min</li>
+<li>25 °C for 10 min</li>
+<li>20 °C for 10 min</li>
+<li>15 °CC for 10 min</li>
+<li>10 °C until further use</li>
 </ul>
+	<li> Transform reaction mixture in <i>E. coli</i> as described in the <a href="#HifiDNAass">Hifi assembly protocol</a></li>
-<h4>Day 3 (mycelial harvest)</h4>
-<ul start="5" class="protocolli">
-	<li>Place the sterile funnel with a mira cloth in to a sterile blue cap bottle and transfer the contents of the shake flasks to the mira cloth (content should be brown and thick).</li>
-	<li>Wash the mycelia using Aspergillus protoplastation buffer (APB) to remove residual glucose from the mycelia (this can inhibit protoplastation). You need to use quite a bit of APB. squeeze out remaining liquid using a sterile spoon. Then, transfer the mycelium to falcon tubes ( ≈2 g per tube => 1 shake flask ≈ 2 falcon tubes). </li>
 </ul>
-<h4> Protoplastation </h4>
-<ul  start="7" class="protocolli">
-	<li>Add glucanex to APB to get a final concentration of 40 mg glucanex per ml APB and dissolve glucanex via gentle magnetic stirring and no heat.</li>
-	<li>Sterile filter 20 ml of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 ml syringe (there is a bit of resistance in the filter but that's ok).</li>
-	<li>Shake/incubate enzyme-mycelium mix at 30 °C, 150 RPM for 2-3 h.</li>
-<li>From now on, whenever you pipette anything with the cells in it, cut of the edge of the pipette tip and CAREFULLY(!) pipette the cells. If you don't do this, they break as they don't have a cell wall to keep them stable.</li>
-<li>Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/ml). Approved protoplast solutions are then diluted by pouring APB up to 40 ml. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile milli-Q H2O and carefully place 5 ml of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)</li>
-<li>In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 ml and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.</li>
-<li>Count protoplasts in microscope by diluting a small sample 1:100.</li>
-<li>Resuspend protoplasts in 4 ml ATB to obtain concentrated solution.</li>
-</ul>
 </div>
-<!--JACOB HERE-->
 <h3> Click below for a lab friendly version of this protocol</h3>
-<a href="#" download="How-to-download-file.pdf">
+<a href="https://static.igem.org/mediawiki/2019/4/40/T--DTU-Denmark--USER_cloning.pdf" download="USER-cloning-protocol.pdf">
      <button class="butn protocolbtn">Download File</button>
 </a>
 </div>
 </div>
-</div>
@@ Line 2,914: / Line 3,181: @@
      </footer>
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+<img class="footergrants" src="https://static.igem.org/mediawiki/2019/9/91/T--DTU-Denmark--biggrants.svg" title="The logos of our three biggest supporters, DTU Blue Dot, Novo Nordisk fonden and Otto Mønsted fonden" alt="The logos of our three biggest supporters, DTU Blue Dot, Novo Nordisk fonden and Otto Mønsted fonden">
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-<img src="https://static.igem.org/mediawiki/2019/d/d9/T--DTU-Denmark--sponsorsfooter.svg" title="The logos of all of our sponsors, DTU, BioNordica, Eurofins Genomics, Qiagen, NEB New England biolabs, IDT Integrated DNA technologies and Twist bioscience" alt="The logos of all of our sponsors, DTU, BioNordica, Eurofins Genomics, Qiagen, NEB New England biolabs, IDT Integrated DNA technologies and Twist bioscience" style="margin: 20px;max-width:100%;margin-right:auto; margin-left:auto;display: block;
-    height:auto;z-index:5;">
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