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Yeast Transformation Protocols

Find more protocols and selection guides in the Molecular Biology Guide.

Introduction

Yeasts are considered model systems for eukaryotic studies as they exhibit fast growth and have dispersed cells. Moreover, replica plating and mutant isolation of yeast cells can be done with relative ease and they have a well-defined genetic system. Most significantly, yeasts have a highly versatile DNA transformation system that can be utilized effectively for protein production.

Yeast Transformation

Table 4: Sigma-Aldrich Yeast Transformation Products
 

Catalog Number Description Application Features/Benefits

YEAST1


Yeast Transformation Kit The product contains:
• 10μg Control yeast plasmid DNA, pRS316
• 100mL PLATE buffer
• 100mL Transformation buffer

Suitable for transformation of any strain of yeast. Convenient, flexible and sensitive, positive transformants can be obtained with as little as 10ng of DNA; the optimum efficiency is in the 0.1-3μg range.

- Easy and ready-to-use
- Requires as little as 10ng of plasmid DNA
- Flexibility for any strain of yeast - Sufficient for over 100 standard transformations


L4158 Lithium acetate dihydrate Facilitate yeast transfection Part of the BioXtra product line
T9285 Tris-EDTA buffer solution TE Buffer contains 1M Tris-HCl (pH ~8.0), containing 0.1M EDTA.
TE (Tris EDTA) Buffer is commonly used in molecular biology labs. TE solubilizes DNA and RNA while protecting it from degradation
Suitable for storage of RNA and DNA, including purified plasmid DNA stocks
D8418 Dimethyl sulfoxide (DMSO)

 



Can be used for buffer preparation for transformation, DEAE-dextran-mediated transfection of cells and cell fusion procedures with 40-50% PEG; among several other applications
- The product can increase transformation efficiency 2 to 5-fold
- This product is free from DNases, RNases, phosphatases and proteases.
D9156 Deoxyribonucleic acid (DNA), single stranded from salmon testes

 

Can be used for yeast transformations

 

This DNA is ethanol precipitated and sonicated to produce single-stranded fragments which co-migrate with the 587 and 831 base pair marker fragments

 

Yeast Transformation Protocol

Protocol Overview

The LiAc transformation method involves three main steps: preparing competent yeast cells, transformation with plasmid DNA, and subsequent plating to select the transformants. Yeast transformants are usually selected using auxotrophic markers; hence, the appropriate synthetic dropout medium is used for screening transformants.

Reagents Required:

Stock Solutions:
50% Polyethylene glycol solution (mol wt ~36,500)
1M Tris-HCl and 0.2M EDTA, pH 8.0 (TE Buffer) (Catalog Number T9285)
1M Lithium acetate, pH 7.5 (Catalog Number L4158)

1x TE-LiAc solution
10 mM Tris-HCl, pH 8.0, with 1mM EDTA and 0.1M lithium acetate

PEG-TE-LiAc solution
40% PEG in 10mM Tris-HCl, pH 8.0, with 1mM EDTA and 0.1 M Lithium actetate

Yeast Transformation

  1. Prepare YPD and synthetic complete (SC) drop-out medium plates and autoclave them separately (see Tables 1 and 2 for Sigma-Aldrich products and refer to "link to yeast media" for media preparation details).
  2. Inoculate yeast cells from plates into 20mL of YPD medium in a 100mL sterile flask.
  3. Grow overnight with shaking.
  4. Dilute cells from above culture into 100mL of YPD medium until the OD600 is 0.3
  5. Pellet cells gently.
  6. Resuspend in 7-8mL of 1x TE-LiAc solution and rotate at 23°C for 1-1.5 hours.
  7. Add 10µL of 10 mg/ml salmon testes DNA (Catalog Number D9156) in sterile microfuge tubes designated for transformation and one for a negative control.
  8. Add 0.1µg of yeast plasmid DNA (to be studied) to each tube and 100µL of competent cells into each tube and then vortex.
  9. Add 600µL of freshly prepared PEG-TE-LiAc solution, vortex, and incubate at 30°C for 30 minutes with shaking.
  10. Optional - DMSO (Catalog Number D8418) can be added to 10% (v/v); followed by heat shock for 15 minutes at 42°C.
  11. Spin for 3 seconds, resuspend cells in sterile water and plate using appropriate SC drop-out medium.

Note: Refer to growth protocols for plating yeasts and the following section for isolating transformants.

Isolation of Yeast Transformants

Yeast transformants are usually selected using the URA3 complementation method, although techniques utilizing amino acids for complementation and blue-white screening methods can also be used.

In the URA3-based selection technique; the plasmid DNA has a normal copy of the yeast URA3 gene, as well as the URA3 promoter; whereas, the yeast mutant strain lacks the URA3 allele. Thus, yeast cells transformed with the plasmid have a functional copy of the URA3 gene, which allows them to grow on Yeast Synthetic Drop-out Medium Supplements without Uracil (Catalog Number Y1501).

 

Figure 1: Yeast Transformation

Materials

     

References

  1. Sherman, F., Getting started with yeast, Methods Enzymol., 350, 3-41 (2002).
  2. Methods in Molecular Biology, Vol. 177, Two-Hybrid Systems: Methods and Protocols, May 2001, Springer Protocols
  3. Current Protocols in Molecular Biology 13.2.1-13.2.12, April 2008, Wiley Interscience
  4. Schiestl and Gietz (1989) Curr. Genetics 16:339-346; Gietz, et al (1995) Yeast 11:355-360
  5. Li, B., and Fields, S., Identification of mutations in p53 that affect its binding to SV40 large T antigen by using the yeast two-hybrid system. The FASEB journal, 7(10), 957-963 (1993).

 

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05/14-1

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