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User:DrJones1935/21 June 2012
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 dH2O 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
