Maheshri:Miniprep

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Alkaline Lysis Miniprep

6/20/06

Narendra Maheshri

Materials

Solution I:

50 mM glucose

25 mM Tris-Cl, pH 8.0

10 mM EDTA

Autoclave and store at 4°C

Alternatively, use sterile components.

Solution II:

0.2 N NaOH

1% (wt/vol) sodium dodecyl sulfate (SDS)

Prepare immediately before use

Ex:

 1N NaOH           2.0 ml           0.2 N
 10% SDS           1.0 ml           1% 
 sterile ddH2O     7.0 ml
 Total:            10.0 ml

Solution III:

 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.

1:1 Phenol:Chloroform

Protocol

Day 1:

Inoculate 2 mL of TB + appropriate antibiotic and grow 8+ hours.

Day 2:

1. Decant culture into 1.7 mL microfuge tube. Spin at 5000 RPM 2” to pellet cells.
2. Completely resuspend culture in 100 uL Solution I, either by vortexing or pipetting. Full resuspension is important for efficient lysis.
3. Add 200 uL solution II (freshly prepared) and invert tubes 4-5 times till the initially cloudy solution clears up.
4. Add 150 uL solution III, and invert tubes 4-5 times. A white precipitate should form containing cell debris and bacterial chromosomal DNA.
5. Spin max speed 5”. Carefully remove supernatant and transfer to a new tube, taking care not to take any debris.
6. Add 200 uL 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.
7. Precipitate DNA by adding 0.7 volumes of isopropanol. Incubate 5” at RT, then spin 5” max speed. A white pellet should form.
8. Decant supernatant and wash pellet with ice-cold 70% ethanol to remove excess salt. Dry pellet.
9. Resuspend pellet in either 50 uL of TE + RNAse or sterile dd H2O + RNAse. (To make RNAse H2O or TE, add 10 uL 10 mg/mL RNAse stock to 990 uL H2O or TE).
10. Incubate 30 minutes at room temperature to allow RNAse to work