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From 2012.igem.org

Make selection plates for Spc, Amp, Kan

• Weigh out 25 g of LB (Miller) medium powder
• Add dH₂O water to 900 mL and allow solids to dissolve fully
• Bring water to 1 L and add 1 mL of 1 N NaOH solution, mix
• Distribute solution evenly into 4x 500-mL E. flasks (250 mL in each) and add 1.5% w/v Bacto-agar (3.75 g/250 mL) to each flask, mix to dissolve solids
• Loosely cover each flask with foil
• Autoclave
• Allow flasks to cool to <60^o C and add the appropriate antibiotic to each solution of LB-agar according to the following dilutions:

Antibiotic	Dilution	Volume added (uL)
Spc	10 uL/mL	2500
Amp	1 uL/mL	250
Kan	0.2 uL/mL	50
LB alone	--	--

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

Make LB broth (1 L) for liquid cultures

• Weigh out 25 g of LB (Miller) medium powder
• Add dH₂O water to 900 mL and allow solids to dissolve fully
• Bring water to 1 L and add 1 mL of 1 N NaOH solution, mix
• Autoclave solution

Inoculate plate culture for Bs strain 1A833 received today

• Place spore-ridden filter disks (3x) of Bs strain 1A833 (Delta MutSL::Spc^r) from BGSC on LB+Spc selection plate
• Rehydrate disks with a few drops of LB broth
• Incubate plate overnight at 37^o C