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From 2012.igem.org

Contents 1 I. Made necessary purchases 2 II. Made antibiotic solutions (use sterile equipment): 2.1 Ampicillin Stock: 2.2 Kanamycin Stock: 3 III. Made plain LB broth 4 IV. Made Ampicillin Agar Plates 5 V. Made Ampicillin and Kanamycin Agar Plates

I. Made necessary purchases

II. Made antibiotic solutions (use sterile equipment):

Ampicillin Stock:

1. Ampicillin – 5 g
2. DI water (sterilized) – 50 mL
3. Dissolve ampicillin in water and pass through a sterilizing filter-syringe system
4. Transfer solution in 1ml increments to sterile Eppendorf tubes
5. Add contents of one eppendorf tube to 1 L of LB broth or agar solution after liquid autoclaving of the agar or broth solutions WHEN they have cooled to about 50C so that one can touch a flask without experiencing pain

Kanamycin Stock:

1. Kanamycin – 2-2.5 g
2. DIwater (sterilized) – 50 mL
3. Same as steps 3-4 for Ampicillin

III. Made plain LB broth

1. Dissolve 25g LB broth or ingredients as indicated in 6/2 protocol in 1 L DI water
2. Autoclave on liquid cycle with water

IV. Made Ampicillin Agar Plates

• 1 eppendorf tube added to cooled but still liquid LB agar

V. Made Ampicillin and Kanamycin Agar Plates

• Contents of 1 eppendorf tube of each antibiotic added to 1 L of agar