TECHNICAL MANUAL

Yeast Kinase YFPFusion Collection
Cat #YSC5108

Product description

Antibiotic resistance

The Dharmacon™ Yeast Kinase YFP Fusion Collection was developed byAnuj Kumar’s research laboratory at the University of Michigan. It is amini-collection of low-copy yeast plasmids bearing gene-YFP fusions. The
genes are cloned along with 1 kb upstream sequence to encompass native
promoters using the Gateway Cloning system. The fusion is at the carboxyterminus of the encoded protein (Figure 1). This plasmid-based collection offluorescent protein fusions is a valuable and versatile resource, facilitating
systematic studies of protein localization (Ma et al. 2008).

Table 1. Antibiotic resistances conveyed by pDEST-vYFP.

B.

BP
reaction

rUR

UR

gene

CEN

Utility

Ampicillin (carbenicillin)

100 μg/mL

Bacterial selection marker

Validation
This collection of Yeast Kinase YFP Fusions was used to investigate thesubcellular distribution of kinases in response to cellular cues, screening fordifferential localization during filamentous growth. Six cytoplasmic kinases(Bcy1p, Fus3p, Ksp1p, Kss1p, Sks1p, and Tpk2p) were identified that localize
predominantly to the nucleus during filamentous growth (Bharucha, Ma etal. 2008).
Open reading frame

attR2
attR2

Clal 28 Hindlll 33

gene

attR1

3000
3000

Origin of replication
Other gene
Notl 596

Promoter

CAT marker

Regulatory sequence
Tag
Unique restriction site

CEN

amp prom

destination vector
attB

promoter

S1

promoter

attR

ccdB
ccdB
destination vector

S1

attB

attB

attR

ARAR

LR
reactionLRreaction

attR

Concentration

vYFP

entryclone

vYFP

entry
clone
attRrpm

8000 8000

attL

genepmGateway
Gatewaycassettecassette(1.7(1.7kb)kb)
Cm r

o ri

promotergene

Cm ro ripromoter

attR1

attP

dBcc

attL

attL

attP

attP

attR1

1/9572dBcc

attL

pDEST-vYFP (9572 bp)

1/9572

ccdB
ccdB
donor vector
donor vector

BPreaction

pDEST-vYFP (9572 bp)

attB

A

attP

attB

A

attB

PCR product

A3

PCR product

A3

A.
attB

Antibiotic

attB
expression
expression clone

lacZ-al Bglll 7756

clone

Figure 1. A suite of destination vectors for recombination-based cloning ofyeast genes as fluorescent protein fusions. A. Overview of Gateway cloning. By
the approach employed here, each amplified PCR product was cloned into the donorvector pDONR221, generating an “entry” clone. A subset of the promoter-gene
cassettes were subsequently introduced into a destination vector, generating an“expression” clone by the LR reaction indicated. The LR reaction is technically simpler
than the initial cloning process; accordingly, the entry clone collection represents
a useful resource for recombination-based subcloning, even without extensive
experience in Gateway-based techniques. B. Plasmid map of the destination vector
pDEST-vYFP, derived from the centromeric yeast shuttle vector YCp50. Arrows indicate
gene-coding sequences.

ccdB other

pBR322 origin
amp marker

attR2 attR2

pDEST-vYFP
10288bp

YFP Reporter

Xhol 7041

Nrul 3316

Kpal 6570
Hpal 6440

URA3 prom
Apal 4378
URA3 marker

Figure 2. Vector map of pDEST-vYFP

dharmacon.horizondiscovery.com

Created using PlasMapper

Protocol I—replication

FAQs/troubleshooting

Table 2. Materials for replication.

ItemVendor

Cat #2X LB-Lennox broth (low salt)

Fisher Scientific

BP1427500

Peptone, granulated, 2 kg - Difco

Fisher ScientificBP9725-2

Yeast Extract, 500 g, granulated

Fisher Scientific

BP1422-500

GlycerolFisher ScientificBP2291

Carbenicillin

Fisher Scientific

BP2648-250

What Clones Are Part Of My Collection?
A USB containing the data for this collection will be shipped with eachcollection. This data file can be downloaded from the Yeast Gene YFPFusion product page: https://horizondiscovery.com/en/gene-modulation/overexpression/non-mammalian/products/yeast-yfp-fusion-kinasecollection
What Antibiotic Should I Use?
You should grow all Yeast Gene YFP Fusion clones in LB-Lennox low saltwith 100 μg/mL carbenicillin.
What Is The Host Strain For This Collection?
The Yeast Gene YFP Fusion clones are in E. coli Top10 Cells (Invitrogen).

2X LB Broth (low salt) Medium Preparation
LB-Broth-Lennox 		Peptone 			Yeast Extract 		

20 g/L
10 g/L
5 g/L

For archive replication, grow all Yeast Kinase YFP Fusion clones at 37 °C
in 2X LB-Lennox (low salt) medium plus 100 μg/mL carbenicillin. Preparemedium with 8% glycerol* and the appropriate antibiotics.

References
1. J. Ma et al., Localization of autophagy-related proteins in yeast usinga versatile plasmid-based resource of fluorescent protein fusions.
Autophagy. 4(6), 792-800 (August 2008).
2. N. Bharucha et al., Analysis of the Yeast Kinome Reveals a Network ofRegulated Protein Localization during Filamentous Growth. Mol. Biol.
Cell. 19(7), 2708-2717 (July 2008).

*Glycerol should be omitted from the medium if you are culturing for plasmid
preparation. If making copies of the constructs for long term storage at –80 °C,
8% glycerol is required.
Freeze at –80 °C for long term storage. Avoid long periods of storage atroom temperature or higher in order to control background recombination
products.

Protocol II—plasmid preparation
For plasmid preparation, grow all Yeast Kinase YFP Fusion clones at 37 °C in
2X LB-Lennox (low salt) medium plus 100 μg/mL carbenicillin. Most plasmidmini-prep kits recommend a culture volume of 1–10 mL for good yield. For
these constructs, 5 mL of culture can be used for one plasmid mini-prep
generally producing 5–10 μg of plasmid DNA.
1. Upon receiving your glycerol stock(s) store at -80 °C until ready to begin.
2. To prepare plasmid DNA first thaw your glycerol stock culture and pulse
vortex to resuspend any E. coli that may have settled to the bottom ofthe tube.
3. Take a 10 μL inoculum from the glycerol stock into 3–5 mL of 2X LB (lowsalt) with 100 μg/mL carbenicillin. Return the glycerol stock(s) to –80 °C.
Note: If a larger culture volume is desired, incubate the 3–5 mL culture
for 8 hours at 37 °C with shaking and use as a starter inoculum. Dilutethe starter culture 1:500-1:1000 into the larger volume.
4. Incubate at 37 °C for 18–19 hours with vigorous shaking.
5. Pellet the 3–5 mL culture and begin preparation of plasmid DNA.
6. Run 3–5 μL of the plasmid DNA on a 1% agarose gel. pDEST-vYFP without
ORF is 10288 bp.

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