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From 2012.igem.org

Contents 1 I. Check Plates and Cultures for Growth 2 II. Make 15% Glycerol Stocks (3 total) from Successful Overnight Culture 3 III. Re-streak B. subtilis (for colonies) and ADP1ΔmutS Plates 4 IV. Make Selective Media for Plasmid Strain Overnight Cultures 5 V. Inoculate Liquid Cultures for Plasmid Strains/CAT* Strain

I. Check Plates and Cultures for Growth

RESULTS (~09:45):

• All plates showed growth except A. baylyi ADP1ΔmutS. ECNR2 plate was sparse, but had colonies. All B. subtilis plates showed robust growth and few individual colonies. Growth was diffuse toward the edges.
• Only the ECFI5 culture and one of the two pIM1463 cultures grew overnight. I suspect that the double inoculation did not work for the pIM1463 cultures, but I am not sure why there was no growth for the pBAV1K-T5-gfp strain. It could be that I chose a colony with a defective plasmid or that there is something wrong with our Kanamycin stock. I have decided to make a glycerol stock of the ECFI5 strain as planned and remake the other overnight cultures.

UPDATE: David may have discovered the problem with the pBAV1K-T5-gfp. I chose a colony that was visibly green, but the documentation with the plasmid says that overproduction of the gfp when the cells reach stationary phase will kill the cells, so it is possible that I pulled a dead colony. To try again, I will choose a colony that is visibly NOT green/glowing under UV.

II. Make 15% Glycerol Stocks (3 total) from Successful Overnight Culture

• Transfer 600 uL of E. coli ECFI5 (CAT*) liquid culture to each microcentrifuge tube (x3)
• Add 200 uL of 60% glycerol to each tube (x3)
• Vortex to mix thoroughly
• Store tubes in the -80 ^oC freezer
• Discard any remaining liquid culture from above cell line

III. Re-streak B. subtilis (for colonies) and 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
B. subtilis 168 (IA1)	LB
B. subtilis PY79 (IA747)	LB
B. subtilis 1A822	LB + Spc
A. baylyi ADP1ΔmutS	LB + Kan (50 ug/mL)

• All plates were somewhat wet with condensation.

• Place A. baylyi plate in the static incubator (~34 ^oC) overnight
• Place B. subtilis plates on the benchtop overnight

IV. 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)

V. Inoculate Liquid Cultures for Plasmid Strains/CAT* Strain

• 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) 18:21, 11 July 2012 (EDT)