McClean:LFM Recipe

Overview

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

(Edit by Emily): I added the actual protocol that is used here - to try and avoid the confusion I experienced:

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

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
   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

Protocol

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

Notes

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

1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. 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.

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

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