"

From 2012.igem.org

I. Check Plates and Cultures for Growth

RESULTS (~09:00):

• No growth on plates (incubator or benchtop) or liquid cultures.

• Shaking incubator somehow was at a temperature above 40 ^oC when I arrived this morning, which may have killed cultures.
• Poor plating technique may be the cause of the B. subtilis failure, but the ADP1ΔmutS strain still has not grown on the Kanamycin plate. I will replate everything and do a parallel streak on LB for the ADP1ΔmutS strain to see if it is really Kan^r.

UPDATE: After changing the temperature on the shaking incubator to once again stabilize at 34 ^oC and allowing the cultures to incubate for the day (~5-6 hours), the pBAV1K-T5-gfp cultures grew turbid. I believe that the 40 ^oC temperature arrested cell growth and that now that the incubator is back at a reasonable temperature, the cultures should grow fine. Considering the plates were getting old and David just streaked out new plasmid strains today, I will wait to make another liquid culture/miniprep until tomorrow.

Also, it is possible that the Kan plates that I was using were too concentrated. I thought they were 10 ug/mL but they were actually 50 ug/mL. I have made more dilute plates and will try plating again tomorrow. ~ Srk3 (talk) 16:07, 12 July 2012 (EDT)

II. Pour plates, selection plates for Kan (12.5 ug/mL)

• Weigh out 12.5 12.50291 g of LB (Miller) medium powder
• Add dH₂O water to 350 mL and allow solids to dissolve fully
• Bring water to 0.5 L and add 0.5 mL of 1 N NaOH solution, mix
• Distribute solution evenly into 2x 500-mL E. flasks (250 mL in each) and add 1.5% w/v Bacto-agar (3.75 3.75025, 3.74982 g/250 mL) to each flask, mix to dissolve solids
• Loosely cover each flask with foil
• Autoclave (Liquid cycle, ~50 min)
• Allow flasks to cool to <60 ^oC and add the appropriate antibiotic to each solution of LB-agar according to the following dilutions:

Antibiotic	Volume added stock	Final Concentration
Kan	62.5 uL (50 mg/mL)	12.5 ug/mL
LB alone	--	--

• Pour 9-10 plates (~25 mL LB-agar soln. per plate) of each
• Allow to cool overnight before use
• Store set plates in sleeve at 4 ^oC for up to 6 months

III. Re-streak B. subtilis Plates

• Using sterilized inoculating loop, scrape top layer of ice of glycerol stock and streak cells (trying to thin better for colonies) 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

• Place B. subtilis plates in the static incubator (~34-35 ^oC) overnight