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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