Experiments

1. Thaw a tube of competent cells from the -80°C freezer on ice for 10 minutes.
2. Add 10μl of DNA solution to the tube and mix gently by flicking.
3. Leave the tube on ice for 30 minutes.
4. Place the tube in a water bath or heating block at 42°C for exactly 45 seconds.
5. Place on ice for 5 minutes.
6. Add 900μl of LB medium at 37°C or room temperature.
7. Incubate at 37°C for 60 minutes in a shaking incubator.

Preparing competent cells
1. Pour the bacterial culture into the prechilled 15-ml glass centrifuge tube on ice.
2. Centrifuge at 7000 rpm at 4 °C, for 5 min.
3. Discard the supernatant.
4. Resuspend the pellet in 1 ml of sterile, ice-cold ddH2O and transfer into a chilled 1.5-ml microcentrifuge tube.
5. Spin in a microcentrifuge at 7000 rpm at 4 °C, for 5 min.
6. Discard the supernatant.
7. Resuspend the pellet in 1 ml of ice-cold ddH2O.
8. Repeat steps 1.5–1.7 two more times for a total of three washes.
9. Spin a final time at 7000 rpm at 4 °C, for 5 min, discard the supernatant, resuspend the pellet in 50 μl of ice-cold ddH2O, and put the cells on ice.
Electroporation
10. Add 50–100 ng of supercoiled plasmid to the electro-competent E. coli and mix gently (do not pipette up and down).
11. Transfer the bacteria to a chilled electroporation cuvette. Be careful to pipette straight in between the metal plates and avoid introducing any bubbles.
12. Cap the cuvette and tap it lightly on the bench to settle the bacteria/DNA mix.
13. Put the cuvette back on ice and carry it to the electroporator.
14. Turn on the electroporator and set it to 1.8 kV, 25 μF, 200 Ω. This is a standard setting for most E. coli strains. Other bacterial strains may require an adjustment of the electroporation conditions.
15. Wipe the cuvette briefly with a Kimwipe to remove any residual water or ice, and then place it in the electroporation chamber.
16. Push the pulse button. The time constant displayed should be around 4 ms.
17. Immediately after the pulse has been delivered, add 1 ml of LB (or other growth medium, e.g., SOC) to the cuvette and pipette quickly but gently up and down. Be aware that the transformation efficiency decreases proportionally to the lag time between the electric pulse and the addition of media.
18. Transfer the mixture to a fresh 1.5-ml microcentrifuge tube.
19. Incubate at 37 °C for 1 h with shaking.
20. Evenly spread 100 μl of your transformation onto a selective plate.

1. Streak the B. subtilis on LB agar plates, incubate overnight at 37 ℃.
2. Pick a single colony, inoculate it in 10 mL of one-step transformation medium and incubate overnight at 37 ℃ in a shaking incubator (180 rpm).
3. After ~18 h, measure the OD450 (typically reach 0.5-1.0), if lower, grow longer time or centrifuge down and resuspend in lower volume of culture supernatant.
4. Transform 1.0 mL of the culture into a sterile Eppendorf tube containing transforming DNA and incubate 30 min at 37 ℃ in a shaking incubator.
5. Plate the medium on a selective agar plate.
6. Incubate the plate overnight at 37 ℃.

1. Transformation of B. subtilis
2. Grow the recipient strain(s) overnight in MGI medium, supplemented with antibiotics if necessary.
3. In the morning, take 500 μl of the overnight culture and inoculate in 2 ml of pre-heated medium MGI.
4. Grow for 4.5 h with shaking.
5. Transfer 250 μl of culture to 1 ml of pre-heated medium MG II and add approximately 1 μg of με plasmid DNA.
6. Grow for 2.5 h with shaking.
7. Plate the cells on selective plates.

2-3 days before the transformation:
1. Grow yeast strain of interest on (YEPhD) agar plate (~2 days). For strains that grow poorly on glucose other carbon sources can be used.
2. Scrape cells from the plate and inoculate ~10 ml YEPhD with the cells.
3. Incubate the cells o/n at 30°C and shaking at 280rpm.
Transformation:
4. Prepare a YEPhD culture (5 ml/transformation) with an OD600 of 0.4.
5. Grow the cells to an OD600 of 1.0-1.5 (2 to 6 h) at 280 rpm at 30°C.
6. Spin down 3 min at 3,000 rpm.
7. Wash the cells twice with 25 ml sterile water.
8. Resuspend the pellet in sterile 0.1 M LiAc (100 µl/transformation) and transport to an eppendorf tube.
9. Spin at 1,300g for 1 min.
10. Resuspend the pellet in 0.1 M LiAc (use 100 µl/transformation) and fill out eppendorf tubes with approximately 100 µl/transformation.
11. Spin at 1,300g for 1 min.
12. Remove the supernatant and add to the dry pellet in this order:
• 240 μl 50% PEG4000
• 35 μl 1 M LiAc
• 75 μl DNA mix (up to 5 μg of DNA + 50 μg [5 μl] of salmon sperm [carrier DNA], with sterile water added to a final volume of 75 μl).
13. Vortex until the pellet is completely resuspended.
14. Incubate 30 min at 30°C.
15. Heat shock 20min at 42°C.
16. Centrifuge tubes at 1,300g for 1 min and discard the supernatant.
17. If an antibiotic resistance marker is introduced, carefully resuspend the cells in 0.5 ml YEPhD and incubate shaking at 30ºC for the time specified in Table 1. If not, continue with step 18.
18. Resuspend the cells carefully in 1 ml sterile water and plate 50 μl onto one selection plate (1/10).
19. Spin down the remaining cells for 1 min at 1,300 rpm.
20. Discard supernatant and resuspend the pellet in 100 μl sterile water and plate onto a second selection plate (9/10).
21. Incubate 2-5 days at 30°C until transformants appear.

1. Prepare sufficient master mix for 10µL reactions (note: scaleable) following this scheme:

Volume	Reagent
2.0 µl	5X Phusion HF buffer
0.8 µl	2.5 mM dNTPs
0.5 µl	Forward primer (10 µM stock)
0.5 µl	Reverse primer (10 µM stock)
0.1 µl	Phusion polymerase
5.1 µl	Purified water
1.0 µl	Template DNA

2. Pipette 10 µl of the mastermix to PCR reaction tubes
3. Perform the following PCR protocol on a thermocycler (note: varies with template/primers):

Step	Temp.	Time
Initial denaturation	98°C	60 sec
Denaturation	98°C	10 sec x 32
Annealing	...°C	30 sec x 32
Elongation	72°C	30 sec/kb x 32
Final elongation	72°C	300 sec
Hold	4°C	Indefinitely

1. Prepare sufficient master mix for 20µL reactions (note: scaleable) following this scheme:

Volume	Reagent
10.0 µl	2x PHIRE Green Hot Start II DNA Polymerase Mix
0.15 µl	Primer Forward 1 (100μM stock)
0.15 µl	Primer Reverse 2 (100μM stock)
9.70 µl	MQ-H2O

2. Pipette 20 µl of the mastermix to PCR reaction tubes
3. Pipette 1 µl of the templates to their respective tubes
4. Perform the following PCR protocol on a thermocycler (note: varies with template/primers):

Step	Temp.	Time
Initial denaturation	95°C	5 min
Denaturation	95°C	10 sec
Annealing	...°C	10 sec
Elongation	72°C	30 sec + 20 sec/kb
Repeat from Denaturation for 30 cycles
Final elongation	72°C	10 min
Hold	12°C	Indefinitely

1. Create the following reaction mixture (50 μl; scale to desired volume):

Amount	Reactant
25 μl	SapphireAmp Fast PCR Master Mix (2X Premix)
To 0.2 μM (final conc.)	Forward Primer
To 0.2 μM (final conc.)	Reverse Primer
0.1-10 ng	Plasmid DNA
To 50 μl	Purified water

2. Perform the following PCR protocol on a thermocycler (note: varies with template/primers):

Temp.	Time
94°C	1 min
98°C	5 sec x 30
..°C	5 sec x 30
72°C	10 sec/kb x 30
12?	Hold?

1. Add 15 μL of MQ-water to a PCR tube.
The following steps are done in a LAF bench.
2. Choose a colony from the agar plate and mark the chosen colony on the backside of the plate and label it the same as on the PCR tube.
3. Take a small pipette tip and gently take up some of the colony (not the entire colony) and transfer it into the MQ water in the PCR tube. Slowly pipette up and down to mix.
4. Incubate the PCR tubes in the thermocycler at 98 °C for 10 min and then store at 12 °C.
While waiting → Prepare the components for the PCR and proceed following a regular PCR protocol.

Use a regular PCR protocol (preferably Phusion) with the mutagenic primers!

1. Mix all of the reagents in the table in a tube

Volume	Reagent
0.5 µl of each insert	DNA insert
1.0 µl	T4 DNA ligase buffer
0.5 µl	T4 DNA ligase (enzyme)
0.5 µl	BSAI restriction enzyme
0.5 µl	BSMBI restriction enzyme
6.5 µl	Purified water (pipette this first)

2. Incubate the mixture in a thermocycler according to the following table:

Step	Temp.	Time (Regular enzymes)	Time (FastDigest)
Initial restriction	37°C	4 min	4 min
Restriction (cycling)	37°C	2 min x 50	1 min x 20-50
Ligation (cycling)	16°C	5 min x 50	2 min x 20-50
Final restriction	37°C	4 min	4 min
Heat inactivation	65°C	10 min	10 min
Hold	12°C	Indefinitely

1. In a PCR tube, mix:

Volume	Reagent
7 μl	Purified water
1 μl	FastDigest Green
1 μl	Template
1 μl	EcoRI

2. Incubate at 37°C for 30 min.

1. Thaw GeneArtTM Gibson Assembly® HiFi Master Mix on ice.
2. Vortex GeneArtTM Gibson Assembly® HiFi Master Mix immediately before use.
3. In a microcentrifuge tube on ice, set up the GeneArtTM Gibson Assembly® cloning reaction as described in the table below:

	1-3 inserts	4-5 inserts	Positive control
Amount of each fragment	0.08 pmol vector, 0.08 pmol each fragment, up to 10 μL combined		10 μL
GeneArt Gibson Assembly HiFi Master Mix	10 μL		10 μL
Nuclease-free water	Fill to 20 μL		-
Incubation time at 50°C	15 min	60 min	15 min

4. Mix the reactions by vortexing, spin down and incubate at 50°C for the recommended time.
5. After incubation, place the reaction mix on ice.

All centrifugations are recommended to be done at 12000g for 1 minute unless specified otherwise.
1. Resuspend pelleted cells in 250μl resuspension solution, mix by pipetting up and down (or vortexing) and transfer to a microcentrifuge tube.
2. Add 250μl lysis solution and mix by inverting 4-6 times (do not vortex). Incubate for 2-3 minutes (not more than 5 minutes).
3. Add 350μl neutralization solution and mix by inverting 4-6 times.
4. Centrifuge for 5 minutes and transfer the supernatant to a spin column.
5. Centrifuge the spin column and discard the flow-through.
6. Add 500μl wash solution (with ethanol).
7. Centrifuge and discard the flow-through.
8. Wash again and centrifuge an additional time.
9. Transfer the spin column to a microcentrifuge tube.
10. Add 50μl elution solution to the center of the spin column membrane without touching it.
11. Incubate for 2 minutes.
12. Centrifuge for 2 minutes and discard the spin column (keep the tube).
13. Make sure the tube is labelled and measure the concentration by Nanodrop.

All centrifugations are recommended to be done at 12000g for 1 minute.
1. Weigh a microcentrifuge tube.
2. Using a scalpel, excise an as small as possible gel slice containing the DNA (avoid UV overexposure). Put the gel slice in the microcentrifuge tube.
3. Weigh (the tube with) the gel.
4. For every 1 mg of gel, add 1 μl of binding buffer to the tube with the gel slice.
5. Incubate the mixture at 60°C until completely dissolved (around 10 minutes). Mix every few minutes by inverting.
6. When the gel is completely dissolved, if it is not yellow, add 10 μl pH 5.2 3 M sodium acetate solution.
7. Vortex briefly and transfer up to 800 μl to a purification column.
8. Centrifuge at 12000g for 1 minute and discard the flow-through.
9. Optional if the DNA will be used for sequencing: Add 100 μl binding buffer, centrifuge and discard the flow-through.
10. Add 700 μl wash buffer (with ethanol), centrifuge and discard the flow-through.
11. Centrifuge again.
12. Transfer the column to a microcentrifuge tube.
13. Add 50μl elution solution to the center of the column membrane without touching it.
14. Centrifuge and discard the column (keep the tube).
15. Make sure the tube is labelled and measure the concentration by Nanodrop.

All centrifugations are recommended to be done at 12000g for 1 minute.
1. To the PCR product, add an equal volume of Binding Buffer and vortex.
2. If the color is not yellow, add 10μl pH 5.2 3M sodium acetate solution.
3. Transfer up to 800μl of the solution to the purification column
4. Centrifuge and discard the flow-through.
5. Add 700μl wash buffer (with ethanol), centrifuge and discard the flow-through.
6. Centrifuge again.
7. Transfer the column to a microcentrifuge tube.
8. Add 50μl elution buffer to the column membrane without touching it.
9. Centrifuge and discard the column (keep the tube).
10. Make sure the tube is labelled and measure the concentration by Nanodrop.

1. Blank the machine by placing 1μL purified water in the notch and pressing “blank”.
2. Wipe the notch dry.
3. Run every sample, wiping after each one.
4. Read the measured concentrations and write them on the tubes.

Preparing agarose solution
1. If not prepared, make an agarose solution by mixing agarose into 1x TAE buffer to a w/w concentration of 0.5% or 1% and microwaving until completely dissolved. Store the solution in 60°C when not in use.
Casting the gel
2. Cast a gel by pouring the solution into a tray of the desired size with a comb of the desired number of wells (including 2 for ladders). The gel should be around 0.5cm in depth.
3. Add stain to the solution (we use GelRed, 1.5μL for 8cm gel) and mix while removing eventual bubbles. Leave the gel to solidify for 30 minutes.
Running the gel
4. Remove the comb and transfer the tray with the gel into an electrophoresis chamber. Make sure the gel is covered in TAE buffer (add more if necessary).
5. Separately mix 1μL of each sample with 4μL purified water and 1μL 6x loading dye.
6. Transfer 3μL of each sample mix into their respective wells, taking care not to touch the gel.
7. Transfer 1.5μL ladder (GeneRuler 1kb) into wells on both sides of the wells with the samples.
8. Close the lid, connect the cables (make sure the polarity is correct) and turn on the machine.
9. Set the machine to the desired voltage and time (our standard: 100V / 30min for 8cm well) and press Run. Look for tiny bubbles near the edges to make sure it’s running.
10. When the run is done, transfer the gel off the tray and onto the machine's gel plate (wipe the gel plate with ethanol first). Place it in the machine and click analyse in the computer program. Edit the contrast etc. for visibility, save the image, discard the gel in the special gel bin and wipe the gel plate with ethanol.

Create a mixture of the desired volume with the following composition (w/w):
• 1% Tryptone
• 1% NaCl
• 0.5% Yeast extract
• Purified water (to desired volume; recommended 500 ml)
Stir the mixture until everything has dissolved and autoclave it (remember not to close the lid too tight).

1. Medium Preparation (500 mL):
2. Dissolve K2HPO4 (9.54 g), KH2PO4 (3.0 g), trisodium citrate dihydrate (0.57 g), MgSO4·7H2O (0.1 g), and Na2SO4 (1.0 g) in ~300 mL dH2O.
3. Adjust to pH 7.0 at room temperature using HCl.
4. Add glucose (2.0 g), sodium L-glutamic acid potassium salt monohydrate (1.38 g), L-tryptophan (20.0 mg), FeCl3 (4.055 mg) and MnSO4·H2O (0.169 mg), mix until fully dissolved.
5. Bring volume to 500 mL with dH2O.
6. Sterilize by sterile filtration and store at room temperature.

• Media composition:
• MGI:

Ammonium sulfate	2 g/l
Na-citrate	1 g/l
Potassium phosphate dibasic trihydrate	14 g/l
Potassium dihydrogen phosphate	6 g/l
Magnesium sulfate	1.6 mM
Caseine hydrolysate	0.25 g/l
Glucose (filter)	5 g/l
Tryptophane (filter)	0.25 g/l

• MGII:

Ammonium sulfate	2 g/l
Na-citrate	1 g/l
Potassium phosphate dibasic trihydrate	14 g/l
Potassium dihydrogen phosphate	6 g/l
Magnesium sulfate	4 mM
Yeast extract	1 g/l
Caseine hydrolysate	0.1 g/l
Calcium nitrate	2.5 mM
Glucose (filter)	5 g/l
Tryptophane (filter)	0.25 g/l

Media should only be exposed to air inside the LAF bench. Components vary with organism.
1. Add 100 μl cell solution to the middle of a plate.
2. Rub around the solution on the plate with a T-spreader until it stops feeling wet.
3. Close the plate (keep plates bottom-up when not in use) and label it (LC) if not done already.
4. Pellet the cells by centrifugation and discard half (or other amount depending on desired concentration) of the supernatant.
5. Repeat steps 1-3 for the new “high concentration” (HC) cell solution.
6. Seal the plates with parafilm and incubate at 37°C shaking overnight.
Plates are stored in the refrigerator when not in use. Tape plates together with colored tape for organization.

Media should only be exposed to air inside the LAF bench. Components vary with organism.
1. Identify the colony (or colonies) to inoculate, preferably isolated colonies.
2. Using the electric pipette, add 3ml (or 5ml) LB medium to a tube.
3. Add the appropriate antibiotic to the tube (amount depends on type).
4. With an inoculation loop, touch the identified colony lightly to pick some (not all) of it up. Hold the loop in the medium and shake it around a bit.
5. Incubate at 37°C shaking overnight.