Smolke:Protocols/Feeding yeast cultures

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Contents

Overview

This protocol is used to set up cultures, feed substrates, and harvest metabolites from media for analysis by LCMS.

Procedure

Materials

  • substrate
  • selective NINR media with dextrose replacing the normal sugars and without water added
  • selective yeast media
  • syringe filter & syringe
  • culture plates
  • Agilent LC plates

Method

Making Substrate Stocks

  • Norlaudanosoline: 20mM stock (5X) (4mM working concentration)
    • Location: Chemical cabinet, labeled (+/-) tetrahydropapaveroline hydrobromide
    • 7.364mg NL into 1mL MQ water.
    • Dissolve by taping to vortexer and let shake for about 10 minutes.
    • Syringe filter.
    • Storage: Make fresh or keep for short amounts of time at -20 or -80 C. Watch out for discoloration of the solution.
  • Dopamine: 100mM stock (10X) (10mM working concentration, can vary up to 100mM)
    • Location: Chemical cabinet, labeled dopamine hydrochloride
    • 18.9mg into 1mL MQ water.
    • Syringe filter.
    • Storage:
  • Tyrosine: 2g/L stock (10X) (0.2 g/L working concentration)
    • Location: Cold room. This is a shared lab stock made by Joe.
    • Make 1-2mL aliquot.
    • Storage: 4C

Growing and Feeding Cultures

Day 1:

  • Set up an overnight culture of strains in selective media (3mL). (Use synthetic complete in place of YPD)

Day 2:

  • Transfer cultures to 96 well shake plate and feed:
    • Norlaudanosoline
      • 400µl appropriate NINR media
      • 100µl 5x NL stock
      • 250µl overnight culture
    • Dopamine and Tyrosine
      • 400µl appropriate NINR media
      • 50µl 10x dopamine stock
      • 50µl 10x tyrosine stock
      • 250µl overnight culture
  • Put into plate shaker. Let grow for 48-72 hours.

Analysis

  • Spin down culture plate on JS-5.3 rotor with appropriate balance at 5000 rpm for 10 min.
    • Make sure plate is well nested on rotor, otherwise it will crack.
  • Transfer 200-300 uL of supernatant to Agilent 96 well round bottom plates. Do NOT disturb the cells. May also clean samples by solid phase extraction or filtration.

References

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