BIO312 Techniques in Molecular Biology

LAB EXPERIMENT 3: Plasmid DNA isolation by BOILING methods

Background & Theory

Four spectroscopic methods are routinely used to determine the concentration of protein in a
solution. Bacteria that contain plasmid DNA are broken open by treatment with lysozyme, Triton
(a nonionic detergent), and heat. The chromosomal DNA remains attached to the bacterial
membrane and is pelleted to the bottom of a centrifuge tube during a brief spin. Plasmid
DNA is precipitated from the supernatant with isopropanol.

This procedure is recommended for preparing small amounts of plasmid DNA from 1 to 24
cultures. It is extremely quick, but the quality of DNA produced is lower than that from the
alkaline lysis miniprep ( coming after this method) .

REFERENCE:

Holmes, D.S. and M. Quigley. 1981. A rapid boiling method for the preparation of
bacterial plasmids. Anal. Biochem. 114, 193-197.

METHOD

Reagents:

STET Buffer 8% sucrose 50 mM TRIS-HCl, pH 8.0 50 mM EDTA 5% Triton X-100

Make up 100 ml STET Buffer at a time. Autoclave.
NOTE: The Triton-X 100 often separates out after autoclaving; therefore, re-mix the
phases well once solution has cooled down.
10 mg/ml lysozyme solution (in TRIS (pH8) buffer ) FRESHLY-MADE
Isopropanol
1 x TE buffer (10 mM Tris, bring to pH 8.0 with HCl and add 1mM EDTA )
LB medium (nutrition for E-coli bacterium with our DNA plasmid pBI101.1- see last
page )
Equipment:

1. Boiling water bath (100C) or heating block

Date: 05.11.2014
Prepared by: Assistant Jasmin Sutkovic
 2. Centrifuge +4C

3. Pipettes

4. 1.5-ml disposable microcentrifuge tubes

Protocol

1. Grow liquid cultures to saturation overnight at 37C in 3-5 mL LB + Kanamycine ( 50 g/ml)

2. Take and Spin down ~ 2 ml of bacterial culture in a 2ml microfuge tube, 20 seconds at
maximum speed.
Note: Heat water bath to boiling (or make sure 100C heating block is on).
3. Resuspend cells in 250 L STET buffer, by gently pipetting up and down (or vortexing).

4. Add 20 L lysozyme solution. Mix briefly by quick vortex or inversion.

5. Immediately place the tubes at 100C for 3 minutes on heating block or water bath .

6. Remove promptly. Let tubes cool down for 5 minutes on bench top.

7. Spin tubes at top speed for 20 minutes.

8. Take the supernatant from pellet slowly with a pipette tip to a new 2ml tube, to separate from
the gooey precipitate/pellet (do not touch the pellet , the pellet should be fairly gummy that
contains bacterial bebris as well as chromosomal DNA).

Note: Supernatant contains DNA/RNA molecules

9. Add 250 L of 100% isopropanol to each tube . Mix well. Put on ice or at 4C for at least 20
minutes (RT is okay, too). If you are in a hurry this incubation can be shortened; however, the
yield might be reduced. Also, this can be a stopping point.

10. Spin down precipitated nucleic acids (DNA+RNA) at top speed for minutes. (The
temperature is not importantpellets will be bigger at 4C, but I think this is mostly due to
salt.)

11. Remove the supernatant and leave the pellet for 3-5 minutes for air dry.

12. Resuspend pellet in 50-200 L TE (let sit at room temperature or on ice for a while).

Date: 05.11.2014
Prepared by: Assistant Jasmin Sutkovic
 13. Use 1-5 L per restriction digest.

NOTE: Don't forget to add RNAse to digests or loading dye, as these preps will have RNA in
them.

DNA- PLASMID (pBI101.1) -13.922bp

Date: 05.11.2014
Prepared by: Assistant Jasmin Sutkovic