McClean:SC Canavanine
Overview
Synthetic complete dropout media (SC-N with the nutrient N dropped out) is the media we use in the lab for selecting strains that have repaired an auxotrophy (e.g. selection on SC-URA plates after transforming with a URA+ plasmid or DNA fragment) or for persistent selection for the ability to grow without nutrient N (e.g. for maintaining a URA+ plasmid, the strain is always grown in SC-URA media).
Synthetic complete is the name given to the media with nothing dropped out (i.e. with all amino acids and other nutrients added the media). You can use synthetic complete for microscopy (it is much better for this purpose than YPD) but low fluorescence media (LFM) is preferable.
Materials
- Yeast Nitrogen Base without Amino Acids (Difco Cat #291940)
- Bacto-agar (Becton-Dickinson #214030)
- 10x Glucose Solution (20% w/v; Sigma D9434)
- 10x Amino acid mix (with appropriate nutrient dropped out)
- Distilled water
Stock Solutions
Canavanine 100mg/mL
Dissolve L-canavanine sulfate salt (Sigma) in water at 100mg/mL, filter-sterilize and store aliquots at 4°C
Thialysine 100mg/mL
Dissolve S-[2-aminoethyl]L-cysteine hydrochloride (Sigma) in sterile miliQ water at 100mg/mL. Filter sterilize using a syringe and store aliquots at 4°C
Protocol
For 1 liter of solid media mix together:
- 6.7g yeast nitrogen base
- 20g bacto-agar
- deionized water to 900ml
Autoclave for 30 minutes on the liquid cycle. Once cooled to about 55°C add 50ml of the 20x glucose solution and 50ml of the 20x amino acid mix. Pour plates.
For 1 liter of liquid media mix together:
- 6.7g yeast nitrogen base
- deionized water to 900ml
Autoclave for 30 minutes on the liquid cycle. Once cooled to about 55°C add 50ml of the 20x glucose solution and 50ml of the 20x amino acid mix.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Burke, D. Dawson, D. & Sterns, T. 2000 Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual Cold Spring Harbor Laboratory Press
Tong and Boone 2006 Synthetic genetic array analysis in Saccharomyces cerevisiae Yeast Protocols
Guthrie, C and G Fink 2004 In vivo mutagenesis and plasmid shuffling Guide to Yeast Genetics and Molecular and Cell Biology Part A
Contact
- Megan N McClean 14:01, 02 November 2011 (EDT)
or instead, discuss this protocol.
