Standard media except with low-fluorescence yeast nitrogen base. (Yeast nitrogen base without riboflavin and folic acid.)
Final Desired Concentration
|5.0 g/L (NH4)2SO4 (or alternate nitrogen source)|
|1.0 g/L KH2PO4|
|0.5 g/L MgSO4|
|0.1 g/L NaCl|
|0.1 g/L CaCl2|
|0.5 mg/mL H3Bo4|
|.04 mg/mL CuSO4|
|0.1 mg/L KI|
|0.2 mg/L FeCl3|
|0.4 mg/L MnSO4|
|0.2 mg/L Na2MoO4|
|0.4 mg/L ZnSO4|
|2 μg/L biotin|
|0.4 mg/L calcium pantothenate|
|2.0 mg/L inositol|
|0.4 mg/L niacin|
|0.2 mg/L PABA|
|0.4 mg/L pyroxidine HCl|
|0.4 mg/L thiamine|
10X Ammonium Sulfate
- 50g (NH4)2SO4; dissolve in ddIH20, bring to 1L total volume, autoclave.
10X Potassium Phosphate
- 10g KH2PO4; dissolve in ddIH20, bring to 1L total volume, autoclave.
100x magnesium sulfate
- 50g MgSO4; dissolve in ddIH20; bring to 1L total volume, autoclave.
- 10g NaCl, dissolve in ddIH20, bring to 500ml total volume, autoclave
- 10g Ca2Cl, dissolve in ddIH20, bring to 500ml total volume, autoclave
- directly from Maitreya Dunham’s chemostat manual
- Metals are made as a 1000X stock that keeps at room temperature for at least a year. Keep the bottle well wrapped in foil since some of the metals are light sensitive. Make the metals in sterile milliQ to avoid contamination. Shake before using since the metals will not totally dissolve.
- Dissolve chemicals in ~1 L milliQ in the following order:
500 mg boric acid (H3BO3) 40 mg copper sulfate.5H2O (CuS04) 100 mg potassium iodide (KI) 200 mg ferric chloride.6H2O (FeCl3) 400 mg manganese sulfate.H2O (MnSO4) 200 mg sodium molybdate.2H2O (Na2MoO4) 400 mg zinc sulfate.7H2O (ZnSO4)
- Taken directly from Maitreya Dunham’s chemostat protocol except we make a 2000X stock and DO NOT use folic acid or riboflavin for low fluorescence media. Substitute sodium pantothenate for calcium pantothenate for calcium free media
- Vitamins are also made as a 2000X stock. The solution is aliquoted into 50 ml Falcon tubes and stored at -20C. Don't fill the tubes to the top, or else the lid will split when frozen. The "working tube" can be stored at 4C. The vitamins will not dissolve completely, so shake before use. Care should be taken to keep the solution well mixed while aliquoting.
2 mg Biotin (Biotin is in the 4C) 400 mg calcium pantothenate 2000 mg inositol (aka myo-inositol) 400 mg niacin (aka nicotinic acid) 200 mg p-aminobenzoic acid 400 mg pyridoxine HCl 400 mg thiamine HCl
Final Composition of Media (1 L total)
- Leave out Glucose if you are planning on making media with Galactose or other alternative carbon sources
|100 ml of 10X nitrogen source (ammonium sulfate or alternative)|
|100 ml of 10X potassium phosphate|
|100 ml of 20% glucose (or Carbon Source)|
|100ml of 10x amino acid supplement|
|10ml of 100X MgSO4|
|5ml of 200x NaCl|
|5ml of 200x Ca2Cl|
|1ml of 1000X metals|
|500 μL of 2000x vitamins|
|578 ml of sterile H20|
For 1 L of LFM:
- Mix 1.7 g LFM powder + 5 g ammonium sulfate + water to 900 mL and autoclave
- Once cooled to ~55 ° C, add 50 mL of 20x glucose solution and 50 mL of appropriate 20x amino acid mix.
LFM powder = YNB (yeast nitrogen base) w/o ammonium sulfate, w/o folic acid, w/o riboflavin
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
*Megan N McClean 12:55, 5 November 2015 (EST) When I originally entered this protocol I incorrectly stated that there was riboflavin in the vitamins (in direct contradiction to this media having riboflavin left out to reduce autofluorescence). I checked my paper notes from 4/20/2010 (MM Lewis-Sigler Ntbk #1 p139). In fact, I did not add riboflavin to the vitamin stock, so the current vitamin stocks are low fluorescence in fact and the recipe should continue to be made without riboflavin. This has been corrected in the protocol above.
Relevant papers and books Adapted from: Sheff MA, Thorn KS. Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast 2004; 21: 661–670 AND Saldanha, AJ et al 2004 Mol Bio Cell AND Maitreya Dunham http://genomics.princeton.edu/dunham/chemostat.html
- Who has experience with this protocol?
or instead, discuss this protocol.