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User:DrJones1935/10 July 2012
From 2012.igem.org
Contents |
I. Streak cells for upcoming transformation with pBAV1K-T5-gfp plasmid
- Using sterilized inoculating loop, scrape top layer of ice of glycerol stock and streak cells onto appropriate plate according to the following table:
| Strain | Plate |
|---|---|
| E. coli ECNR2 | LB |
| A. baylyi ADP1 | LB |
| A. baylyi ADP1ΔmutS::Kanr | LB + Kan (50 ug/mL) |
| B. subtilis 1A833 | LB + Spc |
- Using sterilized inoculating loop, dip into the overnight cultures of the following strains and streak out onto a plate according to the following table:
| Strain | Plate |
|---|---|
| B. subtilis 168 (IA1) | LB |
| B. subtilis PY79 (IA747) | LB* |
- *This plate was wet with condensation. I tried my best to dry it out but it may cause problems.
- Place all plates in the static incubator (~34 oC) overnight
II: Make 15% Glycerol Stocks (6 total) from Successful Overnight Cultures
- Chill a tube of 60% glycerol on ice
- Transfer 600 uL of each liquid culture to each microcentrifuge tube (x3 each)
- B. subtilis 168
- B. subtilis PY79
- Add 200 uL of 60% glycerol to each tube (x3 each)
- Vortex to mix thoroughly
- Store tubes in the -80 oC freezer
- Discard any remaining liquid culture from above cell lines
III. Make Selective Media for Plasmid Strain Overnight Cultures
- Mix:
- For LB + Kan (10 ug/mL):
- 5 mL LB
- 1 uL Kan stock soln. (50 mg/mL)
- For LB + Amp (100 ug/mL):
- 5 mL LB
- 5 uL Amp stock soln. (100 mg/mL)
IV. Inoculate Liquid Cultures for Plasmid Strains/CAT* Strain
- 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 with pBAV1K-T5-gfp (x2 with same colony dip*) | LB + Kan (10 ug/mL) |
| E. coli with pIM1463 (x2 with same colony dip) | LB + Amp (100 ug/mL) |
| E. coli ECFI5 (CAT*) | LB |
- *Made sure to choose colony that appeared green/glowed under UV
- Incubate cultures overnight with shaking at 34 oC
