Rapid disruption of bacterial colonies to test the size of p

Wednesday, June 02, 2004

Description
This method can be use to quickly identify if the correct plasmid (identified here by size) has been transformed into a bacterial cell (generally E. Coli).

Procedure
1: Grow bacterial colonies on rich agar medium (LB or SOC) containing the appropriate antibiotic for your plasmid.

2: Using a sterile toothpick transfer a small amount of a single bacterial colony to a streak or patch on a agar plate with the appropriate antibiotic. Incubate at 37 degrees.

3: Add remainder of colony to 50 ul 10mM EDTA (pH 8.0).

4: Add 50 ul of disruption buffer. Vortex to mix. Incubate at 70 degrees for 5 mins. Allow to cool at room temp.

5: Add 1.5 ul of 4M KCL and 0.5 ul of DNA loading buffer with 0.4% Bromophenol blue. Vortex for 30 secs to mix. Incubate fro 5 mins on ice.

6: Remove bacterial debris by centrifugation at 13,000 rpm for 3-4 mins at 4 degrees.

7: Load 0.7% agarose gel.

8: View bands using a UV transilluminator to identify presence or absence of plasmid.

Recipes
LB agar plates (500ml = 20 plates):2.5 g yeast extract, 5g tryptone, 2.5 g NaCl, 7.5 g Agar (1.5% final conc, make up to 500 ml with distilled water. Autocalve and allow to cool to 50-60 degrees before adding appropriate antibiotic. Mix, pour into plates and allow to cool. Flash with blue flame of bunsen to remove any bubbles.

Disruption buffer: 0.2 N NaOH (fresh), 0.5% SDS, 20 % sucrose.

Supplies
You will require:

Bacterial plates, toothpicks or sterile tips, 1.5 ml eppindorf tubes, centrifuge, vortex and gel electrophoresis apparatus.

Tips
Prior to running clarified SN treat with RNAse to remove RNA band. This will make it easier to see your plasmid DNA band.

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Rapid disruption of bacterial colonies to test the size of p