From 2012.igem.org
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I. Measure OD600 of WT B. subtilis Grown Overnight with 10% Dilution
- Remove 100 uL of saturated culture from 125 mL E-flask
- Mix with 900 uL of plain Nutrient Broth
- Transfer 1 mL of mixture to clean cuvette
- Transfer 1 mL of plain Nutrient Broth to another clean cuvette
- Auto-blank spectrometer with plain NB cuvette
- Read OD600 of culture cuvette
- RESULT: OD600 = 0.13 with 10% dilution, so OD600 of culture is ~1.3
- This result is deemed sufficient to continue, although online protocol recommends OD600 between 2 and 3
II: Make 15% Glycerol Stock (2) of WT B. subtilis culture
- Transfer culture to a clean 15 mL conical tube
- Centrifuge for (5 minutes) 10 minutes at (15000xg) max speed of ~3000 rpm
- Pellet is clearly visible
- Decant supernatant
- Redissolve in 0.875 mL of Nutrient Broth
- Transfer 375 uL of resuspended cells to each microcentrifuge tube (x2)
- Add 125 uL of sterile 60% v/v glycerol stock to each tube (x2)
- Vortex to mix thoroughly
- Flash freeze tubes in liquid nitrogen
- Store in -80 oC freezer
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