McClean:LFM Recipe

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Contents

Overview

Standard media except with low-fluorescence yeast nitrogen base. (Yeast nitrogen base without riboflavin and folic acid.)

Final Desired Concentration

Chemical
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

Stock Solutions

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.

200X NaCl

  • 10g NaCl, dissolve in ddIH20, bring to 500ml total volume, autoclave

200X Ca2Cl

  • 10g Ca2Cl, dissolve in ddIH20, bring to 500ml total volume, autoclave

1000X Metals

  • 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)

2000X Vitamins

  • 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
   200 mg	riboflavin
   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
Chemical
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

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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References

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

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.


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