Reagents Needed:

Lysis Buffer
6.25 ml             2M Tris pH8         25mM
10 ml                 0.5M EDTA         10mM
50 ml                 0.5M Glucose      50mM
433.75 ml          double distilled water
Store refrigerated.

NaOH / SDS
4 ml                   5M NaOH            0.2M
2 ml                   20% SDS            0.2%
94.67 ml            double distilled water
(prepare fresh, DO NOT place on ice)

Potassium Acetate Solution
300 ml              5M KAc
57.5 ml             Glacial Acetic Acid
142.5 ml            double distilled water
Store refrigerated

Phenol:Chloroform:Isoamyl Alcohol (25:24:1) buffer saturated 
Sigma-Aldrich Cat#  P-3803

Chloroform:Isoamyl Alcohol (24:1)
Sigma-Aldrich Cat# C-0549

RNaseA (Ribonuclease A)
Sigma-Aldrich Cat# R-4875

Preamble:
The solutions used in this mini-prep procedure are those of the large scale plasmid prep. In the mini-prep procedure various steps are combined and shortened to speed up the procedure. (µl = microlitre)

Procedure:

• Using a fresh overnight culture of the plasmid containing bacteria, add 1.5 ml of this culture to an eppendorf tube.

• Pellet the bacteria by microcentrifugation for 1 min.

• Aspirate off the culture media.

• Add 200 µl of the LYSIS BUFFER and resuspend the pellet by vortexing. Make sure that the pellet is totally resuspended.

• Add 200 µl of the NaOH / SDS solution and mix by inverting until the solution becomes clear.

• Add 200 µl of POTASSIUM ACETATE solution and vortex until a white percipitate forms.

• Microcentrifuge for 5 min at top speed (>12,000 rpm). Remove the liquid into a fresh eppendorf tube.

• Add 500 µl of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) and vortex vigorously.

• Microcentrifuge for 5 min and then very carefully remove 500 µl of the upper aqueous phase into a fresh eppendorf tube. Be particularly careful NOT to remove any of the organic interphase. Remember..... don't get greedy leave behind 100 µl of the aqueous layer, there is plenty of DNA in the first 500 µl.

• To the aqueous layer add 500 µl of Chloroform:Isoamyl Alcohol (24:1) vortex vigorously, microcentrifuge for 2 minutes and remove 450 µl of the top aqueous layer into a fresh eppendorf tube. (This step will remove any contaminating phenol).

• Percipitate out plasmid DNA from the 450 µl aqueous layer by adding 500 µl of isopropyl alcohol and allowing the sample to sit at room temperature for 5 mins.

• Pellet plasmid DNA by microcentrifugation for 5 mins.

• Aspirate the isopropyl alcohol, recentrifuge briefly and reaspirate any trace amount of alcohol. Air dry the plasmid DNA pellets at room temperature for 20-60 mins.

• Redissolve the plasmid DNA in 50 µl of double distilled water. Add 1 µl RNase A (10mg/ml) to remove residual RNA.

• Quantitate DNA and prepare sequencing samples.