Bridges Lab Protocols

Inositol Labelling of Yeast Cells: Difference between revisions

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fixed some formatting errors
formatted the reagents
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===Methylamine Reagent (make fresh each time)===
===Methylamine Reagent (make fresh each time)===
      5 ml       10 ml
{| border="1"
1.34 ml 2.68 ml 40% methylamine
!  Reagent || per 5mL || per 10 mL || Location
2.29 ml 4.57 ml methanol
|-
0.57 ml 1.14 ml n-Butanol (1-Butanol)
| 40% Methylamine || 1.34 mL || 2.68 mL || Solvent Cabinet
0.805 ml 1.61 ml ddH2O
|-
| Methanol || 2.29 mL || 4.57 mL || Solvent Cabinet
|-
| n-Butanol || 0.57 mL || 1.14 mL || Solvent Cabinet
|-  
| ddH20 || 1.34 mL || 2.68 mL ||
|-  
|}


===n-Butanol-ethyl ether-formate (20:4:1) (make fresh each time)===
===n-Butanol-ethyl ether-formate (20:4:1) (make fresh each time)===
    10 samples
{| border="1"
5 ml n-Butanol (1-Butanol)
!  Reagent || for 10 samples || Location
1 ml ethyl ether  -------(this has an expiration date)
|-
250 l formic acid ethyl ester
| n-Butanol || 5 mL || Solvent Cabinet
|-  
| Ethyl Ether || 1 mL || Solvent Cabinet
|-  
| Ethyl Formate || 0.25 mL || Solvent Cabinet
|}
 


===Inositol-Free Media===
===Inositol-Free Media===
    100 ml  (Enough for 8 samples)
100 mL (enough for 8 samples)
10 ml 10X Amino Acid Stock
{| border="1"
OR Amino Acids –His-Leu-Trp-Ura 10 ml 10X Potassium Salts
!  Reagent || Volume || Location
10 ml NaCl, MgCl2, CaCl2, Soln 10 ml 10X Ammonium Sulfate
|-
1 ml 100X Vitamin Stock 1 ml 100X Trace Elements
| [[10X Amino Acid Stock]] || 10 mL ||
5 ml 40% Glucose (When adding back amino acids Use:  1080 l Uracil (2mg/ml) ;
|-  
53 ml ddH2O 400 uL Tryptophan (10 mg/ml);  864 uL Leucine ; 216 uL Histidine)
| [[NaCl, MgCl2, CaCl2, Solution]] || 10 mL ||
|-
| [[100X Vitamin Stock]] || 1 mL ||
|-
| 40% Glucose || 5 mL ||
|-
| [[10X Potassium Salts]] || 10 mL ||
|-
| [[10X Ammonium Sulfate]] || 10 mL ||
|- 
| [[100X Trace Elements]] || 1 mL ||
|-
| Water || 53 mL
|}
 
When adding back amino acids Use:  1080 uL Uracil (2mg/ml) ; 400 uL Tryptophan (10 mg/ml);  864 uL Leucine ; 216 uL Histidine


==Protocol==
==Protocol==
===DAY1===
===DAY1===
# Grow 10 ml starter cultures at 24C in shaker in the appropriate media
# Grow 10 ml starter cultures at 24C in shaker in the appropriate media
Retrieved from "http://bridgeslab.sph.umich.edu/protocols/index.php/Inositol_Labelling_of_Yeast_Cells"