User:Norman Wang/Alkaline Lysis Plasmid Mini-Prep

Materials

 * E. coli Containing Desired Plasmid(s)
 * Terrific Broth
 * Appropriate Antibiotics
 * Solution 1: GTE Solution
 * 50 mM glucose
 * 10 mM EDTA
 * 25 mM Tris-HCl (pH 8.0)
 * Autoclave mixture and store at 4°C
 * Solution 2: NaOH+SDS Solution
 * 0.2 M NaOH
 * 1% SDS
 * Solution 3: Potassium Acetate Solution
 * 50ml water
 * 29.5ml Acetic Acid, pH to 4.8 using KOH pellets
 * Water to 100ml
 * RNAse A
 * 10 mg/ml
 * 100% Ethanol
 * 100% Isopropanol

Procedure

 * 1) To a 125 mL culture flask, add 6 mL Terrific Broth, 6 uL antibiotics and a colony of bacteria. Include the tip, this helps with aeration. Shake ON at 37°C, 250 rpm.
 * 2) Add 1.5 mL of the culture to 3 eppendorf tubes.
 * 3) Centrifuge at 5,000 rpm for 3 minutes.
 * 4) decant supernatant.
 * 5) add 150 ul Solution 1, resuspend.
 * 6) add 300 ul Solution 2, invert 5 times
 * 7) add 225 ul Solution 3, invert 5 times—very slowly
 * 8) Centrifuge at 12, 000 rpm
 * 9) Combine the supernatant in 1 15ml tube. add 3 ul Rnase A, incubate at 55°C for 1 hour.
 * 10) Add ~1 mL Isopropanol, vortex for 3 seconds, leave in -20°C for 10 minutes
 * 11) Centrifuge at top speed for 15-30 minutes
 * 12) decant supernatant
 * 13) add 3 mL ethanol, with the tip, move the pellet around, incubate at room temperature for 10 minutes
 * 14) centrifuge top speed for 15-30 minutes
 * 15) decant supernatant, remove as much as possible without disturbing the pellet
 * 16) dry the pellet under vacuum. it is not necessary to use the laminar flow hood, you can use the solvent hood. Dry it at 37°C until the pellet is white.
 * 17) add 50 uL TE, place in 37°C to allow plasmids to dissolve. Add supernatant to a small tube for storage.