Lysis by Alkali

From 2011.igem.org

Lysis By Alkali

This protocol is a modification of the methods of Birnboim and Doly (1979) and Ish-Horowicz and Burke (1981).

1. Resuspend the bacterial pellet in 100 uL of ice-cold Solution I by vigorous vortexing.

• It is essential to ensure that the bacterial pellet is completely dispersed in Solution I. This can be achieved rapidly by vortexing two microfuge tubes simultaneously with their bases touching. The original protocol called for the use of lysozyme at this point to digest the bacterial cells. This step is unnecessary. Some strains of bacteria shed into the medium cell-wall components that can inhibit the action of restriction enzymes. This problem can be avoided by resuspending the bacterial cell pellet in 0.5mL of STE (0.1 M NaCl, 10 mM Tris*Cl [pH 8.0], 1mM EDTA [pH 8.0] and recentrifuging. After removal of the STE, resuspend the pellet in Solution I as described above.

2. Add 200 uL of freshly prepared Solution II

• Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Make sure that the entire surface of the tube comes in contact with Solution II. Do not vortex. Store the tube on ice.

3. Add 150 uL of ice-cold Solution III

• Close the tube and vortex it gently in an iverted position for 10 seconds to disperse Solution III through the viscous bacterial lysate. Store the tube on ice for 3-5 minutes.

4. Centrifuge at 12,000g for 5 minutes at 4 degrees C in a microfuge. Transfer the supernatant to a fresh tube.

5. Optional: Add an equal volume of phenol:chloroform. Mix by vortexing. After centrifuging at 12,000g for 2 minutes at 4 degrees C in a microfuge, transfer the supernatant to a fresh tube.

6. Precipitate the double-stranded DNA with 2 volumes of ethanol at room temperature. Mix by vortexing. Allow the mixture to stand for 2 minutes at room temperature.

7. Centrifuge at 12,000g for 5 minutes at 4 degrees C in a microfuge.

8. Remove the supernatant by gentle aspiration. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Remove any drops of fluid adhering to the walls of the tube.

9. Rinse the pellet of double-stranded DNA with 1 mL of 70% ethanol at 4 degrees C. Remove the supernatant as described in step 8, and allow the pellet of nucleic acid to dry in the air for 10 minutes.

10. Redissolve the nucleic acids in 50 uL of TE (pH 8.0) containing DNAase-free pancreatic RNAase (20 ug/mL). Vortex briefly. Store DNA at -20 degrees C.