McClean:SC Media

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

10x Glucose Solution (20% w/v)

For 1 liter of 20% glucose:

• Add 200g Dextrose (Sigma D9434) to 400ml of deionized water with constant stirring. Add the water to the beaker first then sugar! Heat may help the sugar to dissolve.

For 1 liter of 40% glucose (20x concentration):

• Add 400g Dextrose (Sigma D9434) to 800ml of deionized water with constant stirring. Add the water to the beaker first then sugar! Heat may help the sugar to dissolve.
• Once the sugar has dissolved, bring the entire solution up to 1 liter. Divide into smaller bottles (usually you will use 50mls of 40% glucose to make 1L of media, so we usually aliquot about 100mls into small bottles). Autoclave for 30 minutes on the liquid cycle.
• We store stock solutions of 10x glucose by the microwave in Room 115.

10x Amino acid mix

• See KS Amino Acid Supplement

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.

• Megan N McClean 17:32, 2 November 2011 (EDT) Once the bacto-agar and nitroge base have been autoclaved, they can be allowed to solidify. In this state they can be stored for several months. To make plates, microwave the solidified YNB agar, allow to cool to ~55°C in a water bath, add the glucose and amino acids, and pour plates.

References

Burke, D. Dawson, D. & Sterns, T. 2000 Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual Cold Spring Harbor Laboratory Press

Contact

• Megan N McClean 14:01, 02 November 2011 (EDT)

or instead, discuss this protocol.