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.
