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From 2012.igem.org

I: Make 15% Glycerol Stocks (42 total) from Successful Overnight Cultures

• Chill an aliquot of sterile 60% glycerol on ice
• Transfer 600 uL of each liquid culture to each microcentrifuge tube (x3 per culture)

• ECNR2*
• MG1655
• ADP1
• ADP1 ΔmutS (from Dr. Isaacs)
• SIMD41
• SIMD44
• SIMD46
• SIMD47
• SIMD48
• SIMD49
• SIMD58
• SIMD59
• SIMD60
• SIMD61

• *ECNR2 took longer to grow than the other cells and was done separately

• Add 200 uL of 60% glycerol to each tube (x3 per culture)
• Vortex to mix thoroughly
• Store tubes in the -80 ^oC freezer
• Discard any remaining liquid culture from above cell lines

II. Make 100 mg/ mL Ampicillin Stock Solution for Plates/Liquid Cultures

• Weigh out 1 g 0.99942 g of Ampicillin powder from refrigerator
• Vortex with 5 mL of dH_{2O in 15 mL conical tube}
• Bring to 10 mL with dH₂O, vortex
• Wash a clean filter with ~50 mL of sterile water
• Filter sterilize into a clean conical tube, then aliquot 1 mL each to 10 microcentrifuge tubes
• Store in -20 ^oC freezer for up to 1 year, Working concentration = 100 ug/mL

III. Inoculate Liquid Cultures for Cultures Which Failed to Grow Overnight

• Inoculate one isolated colony (or as close as possible) into ~2 mL of appropriate media according to the following table in sterile culture tubes

Strain	Media
E. coli ECNR1	LB
E. coli SIMD40	LB
E. coli SIMD42	LB
E. coli SIMD43	LB
E. coli SIMD45	LB
E. coli SIMD50	LB
E. coli SIMD57	LB + Amp (100 ug/mL)'

• 'For LB+Amp, 14 uL of Amp stock (see above) was added to 14 mL of LB and mixed thoroughly before dispensing

• Incubate cultures overnight with shaking at 37 ^oC