User:Norman Wang/Alkaline Lysis Plasmid Mini-Prep
- 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
- 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.
- Add 1.5 mL of the culture to 3 eppendorf tubes.
- Centrifuge at 5,000 rpm for 3 minutes.
- decant supernatant.
- add 150 ul Solution 1, resuspend.
- add 300 ul Solution 2, invert 5 times
- add 225 ul Solution 3, invert 5 times—very slowly
- Centrifuge at 12, 000 rpm
- Combine the supernatant in 1 15ml tube. add 3 ul Rnase A, incubate at 55°C for 1 hour.
- Add ~1 mL Isopropanol, vortex for 3 seconds, leave in -20°C for 10 minutes
- Centrifuge at top speed for 15-30 minutes
- decant supernatant
- add 3 mL ethanol, with the tip, move the pellet around, incubate at room temperature for 10 minutes
- centrifuge top speed for 15-30 minutes
- decant supernatant, remove as much as possible without disturbing the pellet
- 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.
- add 50 uL TE, place in 37°C to allow plasmids to dissolve. Add supernatant to a small tube for storage.
- Margaret Ruzicka (personal communication) this above protocol gave really good yield on pSB1A3 containing ccdB insert.