McClean:LFM Recipe

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(New page: Standard media except with low-fluorescence yeast nitrogen base. (Yeast nitrogen base without riboflavin and folic acid.) Final Desired Composition of Yeast Nitrogen Base (Note: Ca¬2Cl...)
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[[Category:Template]]
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<!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL!  -->
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==Overview==
 +
Standard media except with low-fluorescence yeast nitrogen base.  
Standard media except with low-fluorescence yeast nitrogen base.  
(Yeast nitrogen base without riboflavin and folic acid.)  
(Yeast nitrogen base without riboflavin and folic acid.)  
-
Final Desired Composition of Yeast Nitrogen Base (Note:  Ca¬2Cl is left completely out for calcium free media; calcium pantothenate is substituted with sodium pantothenate)
 
-
5 g/l (NH4)2SO4  (or alternate nitrogen source)
 
-
1 g/l KH2PO4
 
-
0.5 g/l MgSO4
 
-
0.1 g/l NaCl
 
-
0.1 g/l Ca2Cl
 
-
0.5 mg/l H3BO4
 
-
0.04 mg/l 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 mg/l inositol
 
-
0.4 mg/l niacin
 
-
0.2 mg/l PABA
 
-
0.4 mg/l pyridoxine HCl
 
-
0.4 mg/l thiamine
 
-
 
-
Stock Solutions
 
-
10X Ammonium Sulfate
+
==Final Desired Concentration==
-
50g (NH4)2SO4; dissolve in ddIH20, bring to 1L total volume, autoclave
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{| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse; width:350px" <!--
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This line here formats your table for you.  Change the code to change the formatting of your table.-->
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| align="center" style="background:#f0f0f0;"|'''Chemical'''
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10X Potassium Phosphate
+
<!-- Copy and paste one of the lines above to create a new column in the schedule table.  Alternatively, you can also delete lines to reduce the number of columns.-->
-
10g KH2PO4; dissolve in ddIH20, bring to 1L total volume, autoclave
+
|--
-
100x magnesium sulfate
+
| 5.0 g/L (NH4)2SO4  (or alternate nitrogen source)
-
50g MgSO4; dissolve in ddIH20; bring to 1L total volume, autoclave
+
|--
-
200X NaCl
+
| 1.0 g/L KH2PO4
-
10g NaCl, dissolve in ddIH20, bring to 500ml total volume, autoclave
+
|--
-
200X Ca2Cl
+
|0.5 g/L MgSO4
-
10g Ca2Cl, dissolve in ddIH20, bring to 500ml total volume, autoclave
+
|--
 +
|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
 +
<!-- To add another row to the table copy and paste everything from the |-- line to just above this line.-->
 +
|}
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=='''Stock Solutions'''==
-
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:
+
'''10X Ammonium Sulfate'''
-
+
 
-
500 mg boric acid (H3BO3)
+
* 50g (NH4)2SO4; dissolve in ddIH20, bring to 1L total volume, autoclave.
-
40 mg copper sulfate.5H2O (CuS04)
+
 
-
100 mg potassium iodide (KI)
+
'''10X Potassium Phosphate'''
-
200 mg ferric chloride.6H2O (FeCl3)
+
*10g KH2PO4; dissolve in ddIH20, bring to 1L total volume, autoclave.
-
400 mg manganese sulfate.H2O (MnS)4)
+
 
-
200 mg sodium molybdate.2H2O (Na2MoO4)
+
'''100x magnesium sulfate'''
-
400 mg zinc sulfate.7H2O (ZnSO4)
+
*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 (MnS)4)
 +
  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
   
   
-
Bring total volume to 1 L with milliQ water.
 
-
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)
+
=='''Final Composition of Media (1 L total)'''==
-
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.
+
*Leave out Glucose if you are planning on making media with Galactose or other alternative carbon sources
-
+
{| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse; width:350px" <!--  
-
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
+
-
+
 +
This line here formats your table for you.  Change the code to change the formatting of your table.-->
 +
| align="center" style="background:#f0f0f0;"|'''Chemical'''
-
Final Composition of Media (1L total)
+
<!-- Copy and paste one of the lines above to create a new column in the schedule table.  Alternatively, you can also delete lines to reduce the number of columns.-->
-
100 ml of 10X nitrogen source (ammonium sulfate or alternative)
+
|--
-
100 ml of 10X potassium phosphate
+
| 100 ml of 10X nitrogen source (ammonium sulfate or alternative)
-
100 ml of 20% glucose
+
|--
-
100ml of 10x amino acid supplement  
+
| 100 ml of 10X potassium phosphate
-
10ml of 100X MgSO4
+
|--
-
5ml of 200x NaCl
+
| 100 ml of 20% glucose (or Carbon Source)
-
5ml of 200x Ca2Cl
+
|--
-
1ml of 1000X metals
+
|100ml of 10x amino acid supplement  
-
500 μL of 2000x vitamins
+
|--
-
578 ml of sterile H20
+
|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!
 +
#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 <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
 +
 +
==References==
 +
'''Relevant papers and books'''
Adapted from:  
Adapted from:  
Sheff  MA, Thorn KS. Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast 2004; 21: 661–670
Sheff  MA, Thorn KS. Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast 2004; 21: 661–670
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AND
AND
Maitreya Dunham http://genomics.princeton.edu/dunham/chemostat.html
Maitreya Dunham http://genomics.princeton.edu/dunham/chemostat.html
 +
 +
==Contact==
 +
*Who has experience with this protocol?
 +
 +
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].
 +
 +
<!-- You can tag this protocol with various categories.  See the [[Categories]] page for more information. -->
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 +
<!-- Move the relevant categories above this line to tag your protocol with the label
 +
[[Category:Protocol]]
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[[Category:Media]]
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 +
[[Category:Yeast]]
 +
-->

Revision as of 16:43, 28 June 2011


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 (MnS)4)
  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!

  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.

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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