Bacterial DNA Extraction for PCR

Some polymerase chain reaction (PCR) applications such as gene detection or typing, require little purified DNA and may be performed with crude bacterial extract. Many methods have been described for this procedure and some of them are straightforward and consist simply boiling bacterial cells in water. At this chance, we want to share the extraction method of bacterial DNA for PCR that works well with Gram-positive bacteria such as staphylococci, enterococci, and Clostridium difficile.

Material:

1. Tris-EDTA (TE) buffer: 10 mM Tris-HCl, 1 mM ethylene diamine tetraacetic acid (EDTA), pH 8.
2. Lysis buffer: 50 mM Tris-HCl, 25 mM EDTA, 250 U of lysozyme per milliliter, pH 7.5. Add 15 U of lysostaphine per milliliter for staphylococci.
3. Digestion solution: 0.6% Nonidet P40 (Sigma Chemical, St. Louis, MO), 0.6% Tween 20, 0.6 micrograms of proteinase K per milliliter.

Methods of Bacterial DNA extraction for PCR are:

1. Grow bacteria on appropriate agar or liquid medium.
2. Harvest cells from the culture medium and resuspend them in TE buffer to obtain an optical density of 2.0 at 580 nm.
3. Transfer 1mL of this suspension into a microtube and wash the cells twice with TE buffer by centrifugation (2000g).
4. Resuspend the pellet in 500 microliters of lysis buffer and incubate at 37⁰C for 1 h.
5. Add 500 microliters of digestion solution and incubate at 56⁰C for 1 h.
6. Inactivate proteinase K by heating the microtube at 95⁰C for 10 min.
7. Store the DNA preparation frozen at -20⁰C in aliquots.

The volume of DNA preparation added to the PCR mixture should not exceed 1/20 of the final volume because at a lower ratio, the EDTA present in the preparation may inhibit DNA polymerase activity. The appropriate ratio is dependent on the bacterial species and PCR applications and thus should be optimized.