The selection of plasmids in yeast is based on the use of auxotrophic mutant strains, which cannot grow without a specific medium component (an amino acid, purine, or pyrimidine). Transformation with a plasmid complementing the mutated gene enables the transformant to grow on medium lacking the required component. Yeast cells are made competent for transformation by incubation in a buffered lithium acetate solution. Transformation is then carried out by incubating the cells together with transforming DNA and carrier DNA in a solution containing polyethylene glycol (PEG). The Yeast Transformation Kit utilizes the lithium acetate method that was first introduced by Ito, et al. (1983). The protocols provided include modifications suggested by Hill et al. (1991), Gietz et al. (1992) ( protocol 1), and Elble (1992) (protocol 2).
This kit is sufficient for over 100 standard transformations.
|Transformation Buffer 100 mM lithium acetate, 10 mM Tris-HCl, pH 7.6, and 1 mM EDTA
Product No. T0809
|PLATE Buffer 40% PEG, 100 mM lithium acetate, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA
Product No. P8966
|Deoxyribonucleic acid from salmon testes, 10 mg/mL
Product No. D9156
|2 x 1 ml|
|Control Yeast Plasmid DNA pRS316, carrying the ura gene
Product No. C4959
|Yeast Synthetic Drop-out Medium Supplement without Uracil
Product No. Y1501
This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.
Store the kit at –20 °C. After thawing, store the Transformation Buffer, PLATE Buffer, and Yeast Synthetic Drop-out Medium Supplement Without Uracil at room temperature.
Add 960 mL of deionized water, autoclave for 15 minutes, and add 40 mL of a 50% sterile (0.2 µm filtered)
solution of glucose.
Add 960 mL of deionized water, autoclave for 15 minutes, and add 40 mL of a 50% sterile (0.2 µm filtered) solution of glucose. Autoclaving the yeast-agar solution longer will cause plates to become soft. Alternatively, the medium can be autoclaved longer if the agar is autoclaved separately from the yeast nitrogen base.
Yeast strains can be stored at 4 °C for 2-3 months on YPD or SC plates sealed with Parafilm®. For long term storage scrape a large inoculum from a freshly grown plate and resuspend in 1 mL of sterile 15% glycerol. Store at –70 °C.
To revive frozen yeast stocks, scrape frozen stock with a sterile toothpick or bacteriological needle and streak on the appropriate plate (YPD or SC). Avoid thawing the stock. If the stock has thawed, vortex well before refreezing.
This protocol is more rapid, but less efficient than Protocol 1. It is very convenient when only a small number of transformants are needed, but it is not recommended for transformations of libraries or other precious DNA.
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