Team:DTU-Denmark/Experiments

Experiments

If you've ever participated in iGEM, then you know just how many hours have been spend 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

This protocol is the adapted protocol from 223.

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 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!)
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.

A quickoverview 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 uL DNA ( or max 25% of protoplast volume) and 100 uL 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 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.
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

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.

This guide is to use the biolector.
NB! The machine is very slow - be patient!

Materials

biolector

Procedure

Turn on the machine (bottom in the back, to the right)
Principal window ----> “Monitor”
When the lid opens, place the plate in the machine between the metal barrages - it should be firmly secured.
For create a new program go to second window “Start …” and follow the steps:
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
Select the type of plate (MTP 48 wells FlowerPlate) ----> Next
Choose Layout (48 MTP) ----> Next
Select rpm (1000 rpm), temperature and humidity control, and manual (by defect) ----> Next
Select the filters to use (Biomass –> Gain 10, PFP –> Gain ?, GFP –> Gain ? ) ----> Next
Choose cycle time (30 min for the warm-up program and 3 min (more if several filters are addded) for our program) ----> Start
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.
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.
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.

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
Steril 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 protocolfor 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.

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 1µL of DpnI to finished 50µL PCR reactions (or .5µL to 25µL 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.

Adapted by Jacob Mejlsted & Kyle Rothschild from XXXX (ask Kyle)

Materials

Equipment

Autoclave
Freezer
Centrifuge
Spektrophotometer

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 degC (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 shakeflash around in the cold water to cool culture. Chill the culture in the icewater 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 o 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 CaCl 2 + 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 uL ice-cold 85 mM CaCl2 + 15 % glycerol
Put falcon tubes on ice
Immediately after cells are confirmed resuspended, aliquot 30 uL of the competent cell culture into the chilled PCR tubes
Put tubes into -80 °C freezer as fast as possible

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 transformation

Transformation

[Insert transformation protocol]

Day 2:

Plate counting:

Count CFU
Check control plates
Innoculate LB Cam O/N culture

Day 3:

Miniprep

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 here.

Materials

Reagens

T4 DNA ligase
10X T4 DNA ligase buffer
BsaI HF v2 enzyme (NEB)
Reciever plasmid (BsaI compatible like pGIA2P2o )
DNA fragments with BsaI overhangs
MilliQ water

Media

Materials

PCR tubes
10uL 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
- MilliQ for a total volume of 20 µL.
Mix gently
Place the tube on a thermocycler

Thermocycler program

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

This protocol is made to assembly parts with TypeIIs enzyme SapI

Materials

Reagens

T4 DNA ligase
10X T4 DNA ligase buffer
SapI enzyme (NEB)
Reciever plasmid (Sap one compatible like Inaccessible DNA Sequence )
DNA fragments with SapI overhangs
MilliQ water

Reagens

PCR tubes
10uL 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
- MilliQ for a total volume of 20 µL.
Mix gently
Place the tube on a thermocycler

Thermocycler program

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

Adapted by Jacob Mejlsted & Joen Haahr Jensen from IDT'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
Steril MilliQ water
Competent E. coli
Prepared DNA fragments for assembly (See information on primer construction)

Procedure

Assembly protocol

Set up the following reaction on ice:
* If the inserts are less 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 volumen 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 uL	0.2-0.5 pmols X uL	10 uL
NEB Hifi Assembly master mix	10 uL	10 uL	10 uL
MilliQ water	10-X uL	10-x uL	0 uL
Total volume	20 uL	20 uL	20 uL

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
Note: Extended incubation up to 60 minutes can in some cases improve transformation efficiency

Transformation protocol

Thaw chemically-competent cells on ice
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
Place the mixture on ice for 30 minutes. Do not mix
Heat shock at 42oC for 30 seconds. Do not mix
Transfer tubes to ice for 2 minutes
Add 950 uL of room temperature SOC media to the tubes
Incubate the tube for 37oC for 60 minutes. shake vigerously (250 rpm) or rotate
Warm selection plates to 37oC
Spread 100uL of the cells onto the selection plates.
Note: Use Amp plates for the positive control
Incubate overnight at 37oC.

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 uL
PacI: 2 uL
Cutsmart buffer (NEB): 10 uL
MQ water: 48 uL

For digest II

Finished digest I
Nt.BbvCI: 2uL

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 °Cincubator, add in the 2 uL Nt.BbvCI to the reaction mixture, and incubate for 2 hours at 37 °C
Heat inactivate the samples at 80 °C for 30 min

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 (50uL) [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
MilliQ	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

This protocol is the adapted protocol from 223.

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 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!)
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.

This protocol is the adapted protocol from 223.

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 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!)
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.

This protocol is the adapted protocol from 223.

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 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!)
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.

This protocol is the adapted protocol from 223.

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 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!)
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.

This protocol is the adapted protocol from 223.

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 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!)
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.

This protocol is the adapted protocol from 223.

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 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!)
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.

This protocol is the adapted protocol from 223.

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 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!)
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.

This protocol is the adapted protocol from 223.

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 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!)
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.

This protocol is the adapted protocol from 223.

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 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!)
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.

This protocol is the adapted protocol from 223.

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 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!)
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.

This protocol is the adapted protocol from 223.

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 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!)
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.

This protocol is the adapted protocol from 223.

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 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!)
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.