"

From 2012.igem.org

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