Protocols

Alginate beads production1, 2

The protocol below is solely for making beads that contain cells which are to be used capturing devices: it does not respect sterility rules.

Sodium-alginate cell solution preparation

Estimated bench time: 5 minutes. Estimated total time: 55 minutes.

Purpose: This 2% solution is the basis for the yeast beads.

Materials

2 g alginic acid sodium salt powder (sodium alginate)
100 ml of cell dense media (OD₆₀₀ of 4)
a stirring plate and magnet

Setup and protocol

Mix alginic acid sodium salt powder into 100 ml cell dense media.
Stir for 50 minutes at 700 rpm at 30°C.
Make sure the magnet stays at the bottom of the liquid solution. If not, slow down the rotation until the magnet sinks and slowly resume 700 rpm rotation.

Alternative steps for testing (additional 20-40 minutes)

Since sterility must be guaranteed when testing, first make the sodium alginate solution with 2 g in 67 ml of the media in which your cells grow.
Autoclave at 120°C for 20 minutes.
Mix in 33 ml of yeast solution at OD₆₀₀ of 12. This step might require concentrating the yeast by centrifugation and removal of a part of the supernatant.

Calcium chloride solution preparation

Estimated bench time: 5 minutes. Estimated total time: 5 minutes.

Purpose: This 100 mM solution crosslinks the alginate to form a gel like structure.

Materials

150 ml of distilled sterile water
1.6647 g of CaCl₂

Setup and protocol

Mix CaCl₂ in distilled sterile water.
Stir by shaking gently.

Additional steps for testing

Autoclave for 20 minutes at 120°C.

Bead formation

Estimated bench time: 15 minutes. Estimated total time: 15 minutes.

Purpose: Form regularly sized beads that contain yeast.

Materials

100 ml alginate-sodium-cell solution
250 ml bottle filled with 150 ml of 100 mM CaCl solution
a stirring magnet and a stir plate
a 10 ml syringe (or equivalent)

Setup and protocol (estimated 30 minutes)

Fill the syringe with alginate solution.
Open the CaCl₂ bottle and drop the sterilized magnet inside.
Stir the CaCl₂ solution at 400 rpm during the whole experiment.
Drop drops of alginate into the bottle while stirring.
Continue stirring until 5 minutes have passed since the last bead was made.

Tip: If the alginate is dropped from high enough, interfacial forces between the alginate and the air around it will have enough time to separate a faster stream of liquid into evenly sized spherical beads. This makes the process much quicker.

Bead conservation

Materials

100 ml of media (if cell growth is not desired)
100 ml of deionized water (otherwise)

Setup and protocol

Discard CaCl₂.
Pour the media or water into the bead bottle and keep at 4°C.

Bead dissolution3

Materials

Na-citrate 0.5 M

Protocol

Mix about 10 beads in per 5 ml of 5 0.5 M Na-citrate.
Wait for an approximate 20 minutes for bead dissolution.

Alginate and cell recuperation3

Protocol

Centrifuge the solution at 200g for 20 min.
Recuperate supernatant for reuse.

Bacteria transformation and plating4

Thaw a tube of NEB 5-alpha Competent E. coli: p cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 μl of cells into a transformation tube on ice.
Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
Place the mixture on ice for 2 minutes. Do not mix.
Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
Place on ice for 2 minutes. Do not mix.
Pipette 950 μl of room temperature SOC into the mixture. Immediately spread 50-100 μl onto a selection plate and incubate overnight at 37-42°C. Note: Selection using antibiotics other than ampicillin may require some outgrowth before plating on selective media. Colonies develop faster at temperatures above 37°C, however some constructs may be unstable at elevated temperatures.

Colony picking and culture inoculation

Work under the flame.
With a sterile pipette, scoop up a colony and release the pipette tip into a 5 ml tube containing liquid medium.
Place the solution in the shaker at 30°C.

Copper assay using cuprizone5, 6

Yeast preparation

Purpose: This protocol is a guide for preparing yeasts cultures for copper absorption and removal monitoring.

Materials

appropriate medium (YPD, SGCAA or SDCAA)
CuSO₄ powder
250 ml erlenmeyer
spectrophotometer (OD₆₀₀ measures for yeast population density)
centrifuge

Setup and protocol

Prepare fresh yeast culture in appropriate medium and concentrate it by centrifugation (2000g, 10 minutes, RT) to a density corresponding to an OD₆₀₀ that is 2X the final OD₆₀₀.
Prepare fresh media supplemented with copper by dissolving CuSO₄ powder at a final concentration of 8 mg/l (2X final concentration).
Prepare a separate copper-supplemented medium sample (without yeast) by diluting the 2X copper solution to 1X and keep it as an initial copper concentration control (t_-1).
Start the experiment by mixing half of the yeast culture with half of the copper-supplemented medium in a 250 ml erlenmeyer.
Swirl to assure homogeneity and directly collect 3 times 2 ml of the mix as the first time point (t₀).
Put the erlenmeyers to shaking (220 rpm) at 37°C.
Repeat the collection step at the following time points: t₁ = 15 min, t₂ = 30 min, t₃ = 60 min, t₄ = 90 min and t₅ = 120 min.
Keep collected samples at 4°C until measurement.
Keep the copper-loaded yeasts at 4°C for subsequent copper unloading measurements.

Cuprizone preparation

Purpose: This solution is used for chelating copper, and hence detecting it with a 600 nm absorbance measurement5. Up to 95 tests can be used from the 250 ml produced.

Materials

1 250 ml bottle
1.25 g cuprizone 99%
125 ml deionized water
125 ml ethanol 99%

Setup and protocol

Add cuprizone, water and ethanol into the bottle.
Stir with a magnetic stirrer at 70°C for for 20 minutes or until cuprizone is dissolved.
Let the bottle cool to about 35°C before use.

Tip: The cuprizone solution won't be at the temperature indicated on the stirring plate's screen before a while. Keep the temperature at 200°C during the first 8 minutes for quickly bringing the temperature of the solution to its desired value.

Copper concentration measurement

Materials

media for blanks (450 μl per blank)
samples (450 μl per sample)
cuprizon solution (2.55 ml per sample)
NaOH 0.2 M (3 drops per sample)
pH paper
1 spectrophotometer
4 ml cuvettes (1 per sample)

Setup and protocol

Make the appropriate blanks using the same media as for the samples
Align samples and cuvettes in their respective boxes so that for each cuvette, the position of the corresponding sample is known.
Put 450 μl of each sample into its corresponding cuvette.
Add 3 drops of NaOH 0.2 M.
Set up a timer.
Every 40 seconds, pipette 2.55 ml of cuprizone per cuvette into 3 empty cuvettes until all cuvettes are full. Keep track of the cuvettes as you will have to follow the same order when measuring.
At the 16 minutes time point, measure the first samples. Proceed by measuring 3 samples at a time every 40 seconds.

Building a calibration curve

Purpose: This measurement is needed for calculating the relationship between copper concentration and absorbance. This part can be done simultaneously with the samples.

Warning: Make sure that you use the same blank for the calibration and for the samples and that you use the same media as for the samples.

The calibration curves are made using exactly the same protocol as for the rest of the samples.

Gene PCR7

Thaw, mix and briefly centrifuge each component before use. Avoid generating bubbles when mixing the Master Mix.

Add the following components to each PCR tube. Consider the volumes for all components listed in steps 2 and 3 to determine the correct amount of water required to reach your final reaction volume.

Component	50 - μl rxn	Final concentration
Nuclease-free water	to 50 μl
2X Platinum™ SuperFi™ PCR Master Mix	25 μl	1X

Add your template DNA and primers to each tube for a final reaction volume of 50 μl.

Component	50 - μl rxn	Final concentration
10 μM forward primer	2.5 μl	0.5 μM
10 μM reverse primer	2.5 μl	0.5 μM
Template DNA	varies	varies

Cap each tube, mix, and then briefly centrifuge the contents.

Incubate reactions in a thermal cycler.

Step		Temperature [°C]	Time [s]
Initial denaturation		98	30
30 PCR cycles	Denaturation	98	10
	Annealing	60.4	10
	Extension	72	6.96
Final extension		72	300 (5 min)
Storage		4	∞

Add gel loading buffer and analyze with gel electrophoresis. Add gel loading buffer to 10 μl of PCR product, mix, and briefly centrifuge the contents. Analyze the sample using agarose gel electrophoresis. Use your PCR product immediately in down-stream applications, or store it at -20°C.

Gibson assembly8

Set up the following reaction on ice.

	2-3 fragment assembly	4-6 fragment assembly	Positive control
Total amount of fragments	0.02-0.5 pmols, X μl	0.2-1 pmols, X μl	10 μl
Gibson Assembly Master Mix (2X)	10 μl	10 μl	10 μl
Deinoized H₂O	10-X μl	10-X μl	0 μl
Total volume	20 μl	20 μl	20 μl

Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at -20°C for subsequent transformation.
Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.

Indirect immunofluorescence labeling9, 10, 11

Materials

Potassium phosphate (0.1 M, pH 6.5)
1. Prepare 800 ml of dH₂O in a suitable container.
2. Add 5.874 g of K₂HPO₄ to the solution.
3. Add 9.019 g of KH₂PO₄ to the solution.
4. Add dH₂O until volume is 1 l
P solution
1. 0.1 M potassium phosphate (pH 6.5)
2. 1.2 M sorbitol
This solution is easily contaminated. It is best to prepare a 50 ml aliquot from a 1 l stock.
Zymolyase in P solution (10 mg/ml)
1. Combine 10 mg of dry zymolyase with 1 ml of P solution and vortex.
2. Centrifuge the mixture in a microcentrifuge at 13,000 rpm.
3. Allow the precipitate to settle and use the supernatant.
4. Store this solution at -20°C. It can be used more than once.
PBS-BSA: Phosphate-Buffered Saline containing 5 mg/ml Bovine Serum Albumin.

Protocol

Grow yeast cells in 5 ml of rich or defined media to a concentration of 1 × 107 to 5 × 107 cells/ml (OD₆₀₀ = 1 in a volume of V = 5 ml)
Fix the cells in 4% formaldehyde by adding 0.6 ml of 37% (w/v) formaldehyde directly to the 5 ml culture. Incubate with gentle shaking for 30 min.
Transfer the cells to a 15 ml conical tube and collect them by centrifugation at 800g for 3 min at room temperature.
Aspirate the supernatant and wash the cells with 1 ml of 0.1 M potassium phosphate, pH 6.5.
Collect the cells again by centrifugation at 800g for 3 min at room temperature. Aspirate the supernatant and wash the cells with 1 ml of P solution.
Collect the cells by centrifugation at 800g for 3 min at room temperature. Aspirate the supernatant and resuspend the cells in 1 ml of P solution. Transfer the suspended cells to a microcentrifuge tube.
To digest the cell wall, add 15 μl of 10 mg/ml zymolyase in P solution and 5 μl of β-mercaptoethanol. Incubate the cells with shaking at 30°C until digestion is sufficient.
While the cells are digesting, coat the wells of a multiwell slide with 0.3% poly-L-lysine (this step can be done the day before)
Centrifuge the cells in a microcentrifuge at 800g for 3 min at room temperature. Aspirate and discard the supernatant, and resuspend the pellet in ~ 1 ml of P solution (depending on the desired cell concentration).
Place 25 μl of cell suspension onto each well of a prepared slide. Aspirate the excess cells after several minutes.
Immediately immerse the slide in ice-cold methanol for 6 min. Remove the slide and immerse in ice-cold acetone for 30 sec. Allow the slides to air-dry.
Dilute the primary antibody to an appropriate concentration in PBS-BSA.
Wash the slides once with PBS-BSA (blocking). Aspirate the PBS-BSA and add diluted primary antibody. Incubate the cells in a humidified chamber for at least 2 h.
Dilute the secondary antibody to an appropriate concentration in PBS-BSA.
Aspirate the primary antibody, and wash the wells four times with PBS-BSA. Add diluted secondary antibody to the wells. Incubate in a humidified chamber covered with aluminum foil for at least 2 h.
Aspirate the secondary antibody, and wash the wells three times with PBS-BSA. Wash once with PBS.
Aspirate the PBS and place a drop of anti-fade mounting medium in each well. Place a coverslip on the slide and wipe up the excess mounting medium. Seal the edges with clear nail polish.

Miniprep12

Perform the following procedure at room temperature:

Transfer 600 μl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube.
Add 100 μl of Cell Lysis Buffer, and mix by inverting the tube 6 times.
The solution should change from opaque to clear blue, indicating complete lysis.
Add 350 μl of cold (4 – 8°C) Neutralization Solution, and mix thoroughly by inverting the tube.
The sample will turn yellow when neutralization is complete, and a yellow precipitate will form. Invert the sample an additional 3 times to ensure complete neutralization.
Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
Transfer the supernatant (~ 900 μl) to a PureYield Minicolumn.
Do not disturb the cell debris pellet. For maximum yield, transfer the supernatant with a pipette.
Place the minicolumn into a PureYield Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds.
Discard the flowthrough, and place the minicolumn into the same PureYield Collection Tube.
Add 200 μl of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds. It is not necessary to empty the PureYield Collection Tube.
Add 400 μl of Column Wash Solution to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds.
Transfer the minicolumn to a clean 1.5 ml microcentrifuge tube, then add 30 μl of Elution Buff er directly to the minicolumn matrix. Let stand for 1 minute at room temperature.
Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute the plasmid DNA. Cap the microcentrifuge tube, and store eluted plasmid DNA at – 20°C.

Plasmid digestion13

Set up reaction as follows:

Component	Amount for 50 μl reaction
DNA	1.0 μg
10X rCutSmart Buffer	50 μl (1X)
Restriction enzyme 1 (BamHI)	1 μl (20 units)
Restriction enzyme 1 (BamHI)	1 μl (20 units)
Nuclease-free water	to 50 μl

Incubate at 37°C for 5 – 15 min as both enzymes are Time-Saver qualified.

PCR clean-up14

Gel slice and PCR product preparation

Dissolving the gel slice
1. Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5 ml microcentrifuge tube.
2. Add 10 μl Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50 – 65°C until gel slice is completely dissolved.
Processing PCR amplifications
1. Add an equal volume of Membrane Binding Solution to the PCR amplification.

Binding of DNA

Insert SV Minicolumn into Collection Tube.
Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
Centrifuge at 16,000 g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.

Washing

Add 700 μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
Repeat Step 4 with 500 μl Membrane Wash Solution. Centrifuge at 16,000 g for 5 minutes.
Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.

Elution

Carefully transfer Minicolumn to a clean 1.5 ml microcentrifuge tube.
Add 50 μl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 g for 1 minute.
Discard Minicolumn and store DNA at 4°C or -20°C.

Western blot

Materials

Lysis Buffer: 0.1 M NaOH, 0.05M EDTA, 2% SDS, 2% Beta-mercapto-EtOH (ex: for V = 10 ml, 100 μl of NaOH 10M, 146 mg of EDTA, 1 ml of 20% SDS and 200 μl beta-mercapto, complete with dH₂O)
Loading buffer: 0.25 M Tris-HCl, 50% glycerol, 0.05% Bromophenol Blue (ex: for V = 10 ml, 2.5 ml of 1 M Tri-HCl, 5 ml glycerol and 5 mg bromophenol blue, complete with dH₂O)

Tissue lysis and buffer extraction

Collect 10⁸ yeast cells for each sample (e.g. prepare a solution 1.5 ml of OD₆₀₀ = 1.6 and pellet using centrifuge).
Resuspend in 200 μl lysis buffer.
Boil 10 minutes at 90°C.
Add 5 μl of 4 M acetic acid and vortex.
Boil 10 minutes at 90°C.
Add 50 μl of loading buffer.

Gel run

Preparing the gels: remove plastic, rinse away salts in dH₂O, fill chambers with 2X running buffer.
Load in wells (30 – 40 μl/well for 10 wells/gel). Load four times less 4X loading buffer in empty wells (e.g. 10 μl), same for the ladder.
Run gel 120 V until protein size is low on the gel.
Bend gel using a metal tool to remove the gel from plastic, cut away unnecessary gel.

Blotting

Put the gel in water on the shaker for one minute, then use iBlot for the transfer.

Staining

Wash the membrane with PBS-TX and stain with Ponceau S solution for 10 – 15 minutes.
Cut around where the protein is stained.
Wash with PBS-TX (3x 5 min) and block with fresh PBS-TX 0.1% Milk 5% (e.g. 2.5 g in 50 ml PBS-TX) for 30 min – 1 hour.
Incubate the primary antibody(ies) O/N at 4°C or 1h30-2h at RT.
Wash 3x 5 min with PBS-TX.
Incubate the secondary antibody(ies) for 1 hour at RT.
Wash 3x 5 min with PBS-TX.

Imaging

Prepare the chemiluminescent substrate by mixing each WesternBright Sirius15 solution (2X) together (1 ml each).
Put membrane on the table after drying a little bit on Kleenex.
Dispense the solution homogeneously on the membrane for 5 min.
Dry a little on Kleenex and put in plastic. Turn on Imager and expose.

Stripping (removal of primary and secondary antibodies from the membrane)

If you are planning to keep the membrane for further analyses:

Place the blot in Restore Western Blot Stripping Buffer and incubate for 5 to 15 minutes at 37°C.
Remove the blot from the Restore Western Blot Stripping Buffer and wash with PBS-TX.

Yeast media and plates preparation16

SDCAA

For 1 liter of medium:

Component	Amount
Dextrose	20 g
Difco yeast nitrogen base	6.7 g
Bacto casamino acids	5 g
Na₂HPO₄	5.4 g
NaH₂PO₄	8.56 g

Dissolve all the above ingredients in deionized H₂O to a volume of 1 l and sterilize by filtration. This medium can be stored for up to 6 months at 4°C.

SGCAA

For 1 liter of medium:

Component	Amount
Galactose	20 g
Difco yeast nitrogen base	6.7 g
Bacto casamino acids	5 g
Na₂HPO₄	5.4 g
NaH₂PO₄	8.56 g

Dissolve all the above ingredients in deionized H₂O to a volume of 1 l and sterilize by filtration. This medium can be stored for up to 6 months at 4°C.

Agar plates

Dissolve 5.4 g Na₂HPO₄, 8.56 g NaH₂PO₄·H₂O, 182 g sorbitol and 15 g agar in deionized H₂O to a volume of 900 ml and autoclave. Dissolve 20 g dextrose for SDCAA or galactose for SGCAA, 6.7 g Difco yeast nitrogen base and 5 g bacto casamino acids in deionized H₂O to a volume of 100 ml and sterilize by filtration. Cool autoclaved mixture with stirring until below 50°C, add filter-sterilized solution, mix and pour plates. Plates can be stored for up to 6 months at 4°C.

Yeast transformation17

Preparation of competent cells

Grow yeast cells at 30°C in 10 ml YPD broth until mid-log phase (~ 5 · 10⁶ – 2 · 10⁷ cells/ml or OD₆₀₀ of 0.8 – 1.0). The following steps are accomplished at room temperature.

Pellet the cells at 500 g for 4 minutes and discard the supernatant.
Add 10 ml Frozen-EZ Yeast Solution 1 to wash the pellet. Repellet the cells and discard the supernatant.
Add 1 ml Frozen-EZ Yeast Solution 2 to resuspend the pellet.

Transformation

Mix 50 μl of competent cells with 0.2 – 1 μg DNA (in less than 5 μl volume); add 500 μl Frozen-EZ Yeast Solution 3 and mix thoroughly.
Incubate at 30°C for 45 minutes. Mix vigorously by flicking with finger or vortexing (if appropriate for your DNA) 2 – 3 times during this incubation.
Spread 50 – 150 μl of the above transformation mixture on an appropriate plate. It is unnecessary to pellet and wash the cells before spreading.
Incubate the plates at 30°C for 2 – 4 days to allow for growth of transformants.

References

iGEM TU Eindhoven (2015)

Alginate beads

Full text
AMSBIO

Alginate 3D Cell Culture Kit

Full text
Murujew, Whitton, Kube, Fan, Roddick, Jefferson & Pidou (2019)

Recovery and reuse of alginate in an immobilized algae reactor

Environmental Technology, vol. 42, no. 10, pp. 1521-1530

Crossref Google Scholar
New England Biolabs

5 Minute Transformation Protocol (C2987H/C2987I)

Full text
Messori, Casini, Gabbiani, Sorace, Muniz-Miranda & Zatta (2007)

Unravelling the chemical nature of copper cuprizone

Dalton Transactions, no. 21, pp. 2112

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Soares, Costa, Araujo, Teixeira & Dantas (2018)

Comparison of Spectrophotometric Methods for the Determination of Copper in Sugar Cane Spirit

Journal of AOAC INTERNATIONAL, vol. 101, no. 3, pp. 876-882

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ThermoFischer Scientific

Invitrogen Platinum™ SuperFi™ PCR Master Mix

Full text
New England Biolabs

Gibson Assembly® Protocol (E5510)

Full text
Silver (2009)

Indirect Immunofluorescence Labeling in the Yeast Saccharomyces cerevisiae

Cold Spring Harbor Protocols, vol. 2009, no. 11, pp. pdb.prot5317

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Corbett Laboratory

Immunofluorescence

Full text
Pemberton (2014)

Preparation of Yeast Cells for Live-Cell Imaging and Indirect Immunofluorescence

Methods in Molecular Biology, pp. 79-90

Crossref Google Scholar
Promega

PureYield™ Plasmid Miniprep System

Full text
New England Biolabs

NEB Cloner Double Digestion

Full text
Promega

Wizard® SV Gel and PCR Clean-Up System

Full text
Advansta

WesternBright Sirius HRP substrate

Full text
Chao, Lau, Hackel, Sazinsky, Lippow & Wittrup (2006)

Isolating and engineering human antibodies using yeast surface display

Nature Protocols, vol. 1, no. 2, pp. 755-768

Crossref Google Scholar
Zymo Research

Frozen-EZ Yeast Transformation II Kit

Full text

Team:EPFL/Protocols

Protocols

Alginate beads production1, 2

Sodium-alginate cell solution preparation

Materials

Setup and protocol

Alternative steps for testing (additional 20-40 minutes)

Calcium chloride solution preparation

Materials

Setup and protocol

Additional steps for testing

Bead formation

Materials

Setup and protocol (estimated 30 minutes)

Bead conservation

Materials

Setup and protocol

Bead dissolution3

Materials

Protocol

Alginate and cell recuperation3

Protocol

Bacteria transformation and plating4

Colony picking and culture inoculation

Copper assay using cuprizone5, 6

Yeast preparation

Materials

Setup and protocol

Cuprizone preparation

Materials

Setup and protocol

Copper concentration measurement

Materials

Setup and protocol

Building a calibration curve

Gene PCR7

Gibson assembly8

Indirect immunofluorescence labeling9, 10, 11

Materials

Protocol

Miniprep12

Plasmid digestion13

PCR clean-up14

Gel slice and PCR product preparation

Binding of DNA

Washing

Elution

Western blot

Materials

Tissue lysis and buffer extraction

Gel run

Blotting

Staining

Imaging

Stripping (removal of primary and secondary antibodies from the membrane)

Yeast media and plates preparation16

SDCAA

SGCAA

Agar plates

Yeast transformation17

Preparation of competent cells

Transformation

References