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From 2012.igem.org

Contents 1 I. Check Plates for Growth 2 II. Re-streak ADP1ΔmutS Plates 3 III. Make Selective Media for Plasmid Strain Overnight Cultures 4 IV. Inoculate Liquid Cultures for Plasmid Strains

I. Check Plates for Growth

• RESULTS: All overnight plates of B. subtilis showed growth, including the plates from two days ago left on the bench top. I have saved (on the benchtop) the best looking plate for each of the three B. subtilis cell lines, cataloged in the following table:

Strain	Date of Plating (from...)
B. subtilis 168	11 Jul 12 (bench)
B. subtilis PY79	11 Jul 12 (bench)
B. subtilis 1A833	12 Jul 12 (inc.)

• Also, the E. coli ECNR2 strain plate left on the bench top has continued to grow, producing more viable colonies.

Spencer Notebook 6.JPG B. subtilis PY79, 1A833, 168 (as of 13 Jul 12)

Spencer Notebook 7.JPG A. baylyi ADP1 (wt) and E. coli ECNR2 (as of 13 Jul 12)

II. Re-streak ADP1ΔmutS Plates

• 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
A. baylyi ADP1ΔmutS	LB
A. baylyi ADP1ΔmutS	LB + Kan (12.5 ug/mL)

• Place A. baylyi plates in the static incubator (~34 ^oC) overnight

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

• Inoculate one isolated colony (or as close as possible) into 5 mL of appropriate media according to the following table in sterile E-flasks

Strain	Media
E. coli with pBAV1K-T5-gfp*	LB + Kan (10 ug/mL)
E. coli with pIM1463	LB + Amp (100 ug/mL)

• *Made sure to choose colony that DID NOT appear green/glow under UV

• Incubate cultures overnight with shaking at 34 ^oC

• Completed ~ Srk3 (talk) 18:31, 13 July 2012 (EDT)