Lidstrom:Measuring 13C Incorporation Into Protein - CO2 Project
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- Grow 1.5 mL of OD550 0.8 - 1.5 (approximately 0.3 - 0.8 mg) of cell culture
- Swirl 30-40 seconds in ethanol dry ice bath or liquid N2 (don't freeze sample with liquid N2)
- Centrifuge 5 min at 13,500 g, 4oC
- Discard supernatant
- Wash pellet with 0.9% 1 mL of sodium chloride at 4oC, pellet for 5 min at 13,500 g, 4oC
- Repeat last step
- store pellet at -40 to -80oC
Hydrolysis of Amino Acids
All steps in this section should be done in the fume hood across from the GC:
- Suspend cell pellets in 1 mL of 6N HCl
- Transfer resuspended cells into 2 mL GC-MS autosampler vials
- Seal tubes with screw caps to prevent evaporation of HCl
- Bake the well-sealed 2 mL tubes for 12-24 hours in a heating block set to 105 - 110 (not hotter)oC
- Hotter temps will may destroy some amino acids
- Dry the hydrolysate at 95oC with constant air flow (or N2) gas flow in the fume hood until the sample is completely dry.
- Use the "nitrogen tree"
- Bake the dried samples at 105oC for another 10 minutes to ensure no moisture is left.
Derivitization of Amino Acids
- Add 200 uL nanopure water to reconstitute the dried sample
- If you have a lot of biomass, add 100 uL instead
- Centrifuge at 13,500 g for another 1 - 2 minutes to remove ash
- Use a filter centrifuge tube if sampling a large fraction of this volume
- If using a very small volume (~ 10 uL) you can pellet without a filter and sample from the top of the supernatant. Centrifuge longer (10 min) if using this approach.
- Transfer into clean GC-MS vial with insert, and repeat drying step with nitrogen tree as done above or speed-vac
- Yanfen uses 10 uL; we should use 20 uL
- We can prepare 40 uL + samples to use if the signal from 20 uL is too low
- If using speed-vac: 35oC for 1-2 hours until dry. Hold vials in 15 mL tubes.
- Prepare pyridine:
- Add one layer of molecular sieve to scintillation vial
- Add 1 mL pyridine per sample: found in Hackett lab cabinet under fume hood next to FPLC fridge.
- Let sit for 5 min. No agitation.
For each sample:
- Add 20 uL of molecular-sieve treated pyridine to the dried sample using a syringe (would dissolve pipette tip)
- The solvent may turn slightly brown
- Add another 20 uL of Trifluoromethanesulfonic acid tert-butyldimethylsilyl ester (TBDMS) and seal well
- use the same blue screw-cap but replace the septum
- Rinse syringe in leftover pyridine
Repeat for all samples