TECHNICAL MANUAL

Yeast barcoders
Cat. #YSC5117

Product description
The Yeast Barcoders Collection was developed by Corey Nislow and GuriGiaever at the University of Toronto. The goal of this collection was to provide
a simple and general method of barcoding yeast collections. The YeastBarcoders Collection contains unique DNA sequence tags (barcodes) thatcan be systematically transferred to any Saccharomyces cerevisiae collection(Figure 1). The Yeast Barcoders Collection currently consists of approximately

A.

UKanMX4

1200 yeast strains that combines simple barcoding (Yan, Costanzo et al. 2008)
with Synthetic Genetic Array (SGA sites.utoronto.ca/boonelab/sgatechnology/index.shtml;) (Tong and Boone 2006; Boone 2007).
Constructs are distributed as cultures of S. cerevisiae in YPD broth + G418 (300μg/mL) + 15% glycerol.

DB.
HO

Gene X

Gene X NAT
XGene XHO
Select diploid strain

BarcoderCollection

Select barcoded gene X::DAmpstrain

SGA
Gene X NATGene XHO

Gene X NAT

Figure 1. Strategy used to bar code DAmP strains. A. Two unique bar codes, upstream tag (U) and downstream tag (D), were linked to the KanMX4 cassette.
B. The bar-coded KanMX4 cassette was used to replace HO in the MATa strain BY4741 by transformation and homologous recombination. A bar-coded BY4741 strain was then matedwith a strain f MATa gene X::DAmP strain, and the diploid strains were selected on YPD medium containing G418 and nourseothricin. The diploid strains were then sporulated, and theSGA technology was used to select haploid double mutants that contain both the bar-coded HO and the gene X::DAmP allele. NAT represents the gene that encodes resistance to theantibiotic nourseothricin (Yan, Costanzo et al. 2008).

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Strain information

Replication of 96-well plates

Genotype of S. Cerevisiae BY4741
MATa his31 leu20 ura30 met15D0

Prepare target plates
1. Prepare deep well 96-well target plates by dispensing 1.5mL media withappropriate antibiotics.

Antibiotic resistance
The Yeast Barcoders contain the kanR gene which should be selected for with
G418 (Table 1).
Table 1. Antibiotic resistance of S. cerevisiae yeast strain R1158.

AntibioticConcentration

UtilityG418

300 μg/mL

Eukaryotic selection markerValidation
Construct validation
To verify correct integration of the barcode cassettes, genomic DNA wasprepared from the resistant strains and used as template in PCR reactions
using two primers common to the KanMX4 module (Yan, Costanzo et al. 2008).
Collection validation
To validate both the barcoders the technology was applied by generating acollection of barcoded ‘decreased abundance by mRNA perturbation’ (DAmP)
loss-of function strains. (Yan, Costanzo et al. 2008).

Protocol I – replication
We recommend making a stock or working culture of the yeast strains. Grow
the yeast strains in YPD broth with the appropriate antibiotic (300 μg/mlG418)*. Transfer 850 μL of culture into a polypropylene tube and add 150 μL
sterile glycerol to make a 15% glycerol freezing solution. Vortex the culture
to evenly mix the glycerol throughout the culture. The culture can be storedindefinitely at -80 °C.
Table 2. Materials for yeast replication.

ItemVendor

Cat #Yeast Extract, 500 g, granulated

Fisher Scientific

BP1422-500

Peptone, granulated, 2 kg – Difco

Fisher ScientificBP9725-2Glucose (D (+) – glucose monohydrate)

EMD Millipore

1.08342.2500

GlycerolFisher ScientificBP2291

G418

Calbiochem

34581096-well microplates

Nunc

260860

Aluminum seals

Nunc

276014

Disposable replicators

Genetix

X5054*When preparing media for yeast clones, do not add the glycerol to the medium
until after the clones have grown. Glycerol inhibits the growth of yeast

YPD medium 1 liter
YPD Mix:
Yeast extract
10 g
Peptone 		20 g
dH2O 		900 mL
Autoclave mixture for 20 minutes at 121 °C
Glucose/dH2O mix:
Dextrose 		
20 gdH2O 		100 mL
Shake until in solution and autoclave for 20 minutes at 121 °C. Under hood,
add YPD mix to 100 mL of sterile glucose/dH2O mix.

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Prepare source plates
1. Remove the lids and the aluminum seal from the source plates. Removing theseals while the source plates are frozen will minimize cross-contamination.
2. Allow the source plates to thaw completely with the lids on. Wipe any
condensation that may appear under the lids with ethanol andan absorbent wipe.
Replicate
1. Gently place a sterile, disposable replicating tool into the source plate andlightly mix the yeast cells. Make sure to scrape the bottom of each wellthoroughly ensuring maximum transfer of cells.
2. Gently remove the replicating tool from the source plate and gently insert thetool into the target plate. Mix the replicating tool around in the target plate.
3. Dispose of the plastic replicating tool.
4. Replace the lid of the target plate and the source plate.
5. Repeat steps 1-6 until all plates have been replicated.
6. Heat seal source plates and return to an ultralow freezer.
7. Cover with a microporous film and place the target plates on a 30 °C incubatorwith shaking for at 16-48 hours, based upon when growth is apparent.
8. When sufficient growth has been noted in the target plates, add 500 µL of 50%
glycerol to each well for a final concentration of 12.5% glycerol.
9. Heat seal target plates and return to an ultralow freezer.

Protocol II – strain verification
PCR can be used to verify the integration of the barcode cassette. Prepare
genomic DNA from the resistant strains and use as template in PCR
reactions using two primers common to the KanMX4 module. [KanB(5’-CTGCAGCGAGGAGCCGTAAT-3’) and KanC (5’-TGATTTTGATGACGAGCGTAAT-3’)]and two primers [A (5’-TATTAGGTGTGAAACCACGAAAAGT-3’) and D(5’-CATGTCTTCTCGTTAAGACTGCAT-3’)] flanking HO gene. Primer A is designed
from region 244 base upstream of the start codon, whereas primer D is 356 bases
downstream of the stop codon. For correct transformation strains, the A-KanB andthe D-KanC PCR reactions should produce a 566 and 1964-bp band respectively
when analyzed by gel electrophoresis. To further confirm correct integration ofbarcodes, two other primers [B (5’-ACTGTCATTGGGAATGTCTTATGAT-3’) and C(5’-GAGTGGTAAAAATCGAGTATGTGCT-3’)] were designed based on the sequence
within the HO gene. The A-B and C-D PCR reactions should produce no band if
integration is correct (Yan, Costanzo et al. 2008).

Related reagents
Table 3. Related Reagents.

ItemVendor

Cat #Yeast Barcoders Collection

Dharmacon

YSC5117

DAmp Library for 970 essentialgenes – diploid

Dharmacon

YSC5050

DAmP Library for 842 essential
genes – haploid

Dharmacon

YSC5090

DAmP individual strain – diploid

Dharmacon

YSC5093

DAmP individual strain – haploid

Dharmacon

YSC5094

FAQS/troubleshooting

References

For answers to questions that are not addressed here, please email technicalsupport ts.dharmacon@horizondiscovery.com with your question, your sales
order or purchase order number and the catalog number or clone ID of theconstruct or collection with which you are having trouble.

1. Costanzo, M., G. Giaever, et al. (2006). Experimental approaches to identify
genetic networks. Curr Opin Biotechnol. 17(5): 472-80.
2. Boone, A. T. a. C. (2007). High-throughput strain construction and systematicsynthetic lethal screening in Saccharomyces cerevisiae.
Yeast Gene Analysis. 36: 369-386, 706-707.
3. Tong, A. H. and C. Boone (2006). Synthetic genetic array analysis in
Saccharomyces cerevisiae. Methods Mol Biol. 313: 171-92.
4. Yan, Z., M. Costanzo, et al. (2008). Yeast Barcoders: a chemogenomic
application of a universal donor-strain collection carrying bar-code
identifiers. Nat Methods 5(8), 719-725 (August 2008).

What clones are part of my collection?
A USB containing the data for this collection will be shipped with eachcollection.
What antibiotic should I use?
You should grow all Yeast Barcoders Clones in YPD broth + G418 (300 μg/mL). When preparing medium for yeast clones, do not add the glycerol to themedium until after the clones have grown if a freezer stock is needed. Glycerol
inhibits the growth of yeast. We prepare a solution of 50% glycerol and 50%
medium to add to the growth media after incubation.
Where can I find more information on the barcode primers—such assequences?
The supplemental data included in the Yan 2008 reference contains primer
sequences for the barcodes.
What Is SGA?
SGA stands for the Synthetic Genetic Array developed by the Boone lab at theDonnelly Centre for Cellular and Biochemical Research, University of Toronto(Tong and Boone 2006; Boone 2007).

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