Alkaline Lysis Miniprep
50 mM glucose
25 mM Tris-Cl, pH 8.0
10 mM EDTA
Autoclave and store at 4°C
Alternatively, use sterile components.
0.2 N NaOH
1% (wt/vol) sodium dodecyl sulfate (SDS)
Prepare immediately before use
1N NaOH 2.0 ml 0.2 N 10% SDS 1.0 ml 1% sterile ddH2O 7.0 ml Total: 10.0 ml
5 M KOAc sterile 60 ml glacial acetic acid 11.5 ml sterile ddH2O 28.5 ml Total: 100 ml
The resulting solution is 3 M potassium and 5 M acetate and has a pH of about 4.8.
RNAase (10 mg/ml):
Dissolve 100 mg RNAase A (pancreatic RNAase) in 10 ml 10mM Tris-HCl 15 mM NaCl. Heat to 100 degrees C (in a beaker of boiling water) for 15 minutes. Cool slowly to room temperature. Dispense into aliquots and store at -20 degrees C.
Inoculate 2 mL of TB + appropriate antibiotic and grow 8+ hours.
- Decant culture into 1.7 mL microfuge tube. Spin at 5000 RPM 2” to pellet cells.
- Completely resuspend culture in 100 μL Solution I, either by vortexing or pipetting. Full resuspension is important for efficient lysis.
- Add 200 μL solution II (freshly prepared) and invert tubes 4-5 times till the initially cloudy solution clears up.
- Add 150 μL solution III, and invert tubes 4-5 times. A white precipitate should form containing cell debris and bacterial chromosomal DNA.
- Spin max speed 5”. Carefully remove supernatant and transfer to a new tube, taking care not to take any debris (which contains genomic DNA).
- Add 200 μL 1:1 Phenol:Chloroform and vigorously shake tubes. The phenol:chloroform is used to extract proteins which migrate to the interface between the organic and aqueous layers. Spin tubes at max speed for 2”. Carefully remove aqueous layer (on top) taking care not the disturb the interface.
- Precipitate DNA by adding 0.7 volumes of isopropanol. Incubate 5” at RT, then spin 5” max speed. A white pellet should form.
- Decant supernatant and wash pellet with ice-cold 70% ethanol to remove excess salt. Dry pellet.
- Resuspend pellet in either 50 μL of TE + RNAse or sterile dd H2O + RNAse. (To make RNAse H2O or TE, add 10 μL 10 mg/mL RNAse stock to 990 μL H2O or TE).
- Incubate 30 minutes at room temperature to allow RNAse to work
- Alternatively, one can add 1μL 10 mg/mL RNAse to the supernatant obtained after step 4 and incubate at 37°C for 30 min, before phenol/chloroform extraction. The final DNA pellet can be resuspended in just TE or ddH2O.