Streptomyces DNA isolation

Streptomyces DNA Isolation

From 5 mL of culture

Materials

• 50/20 TE buffer (50 mM Tris, 20 mM EDTA)
• Lysozyme (5 mg/mL)
• RNase (0.1 mg/mL)
• 10% SDS
• Proteinase K (20 mg/mL)
• 5 M NaCl
• Phenol:chloroform:isoamyl alcohol (25:24:1)
• Chloroform:isoamyl alcohol (24:1)
• Isopropanol
• 70% ethanol (cold)

Method

1. Suspend cells in 300 µL 50/20 TE with 5 mg/mL lysozyme and 0.1 mg/mL RNase. Incubate 30 min at 30–37 °C.
2. Add 50 µL 10% SDS and mix thoroughly.
3. Add 10 µL of 20 mg/mL proteinase K and mix. Incubate at 55 °C for 2.5 h.

  *This step is optional, but can reduce DNase activity. Incubation time can be varied.*

1. Add 85 µL 5 M NaCl.
2. Add 400 µL phenol:chloroform:isoamyl alcohol (25:24:1). Vortex 30 s. Centrifuge 10 min at ≥8000 rpm.
3. Transfer the aqueous (top) phase to a new tube, avoiding the organic layer.
4. Add 400 µL chloroform:isoamyl alcohol (24:1). Vortex 30 s. Centrifuge 10 min at ≥8000 rpm.
5. Transfer the aqueous (top) phase to a new tube.
6. Add 0.5 mL isopropanol. Mix by inversion and incubate 5 min at room temperature.
7. Spool out DNA or centrifuge at 10,000 rpm to pellet DNA.
8. Wash pellet with 1 mL cold 70% ethanol.
9. Decant ethanol and allow DNA to air-dry 30–60 min.

  *Do not overdry — DNA will be difficult to dissolve.*

1. Resuspend DNA in TE (10/0.1) or water.

Notes

• **TE buffer (Tris-EDTA):** Solubilizes DNA and protects it from degradation. EDTA chelates metal ions (e.g., Mg²⁺).
• **Lysozyme:** Breaks down bacterial cell walls by hydrolyzing peptidoglycan.
• **Proteinase K:** Digests proteins, including nucleases that degrade DNA.
• **SDS (sodium dodecyl sulfate):** A detergent that denatures proteins by disrupting non-covalent bonds.
• **NaCl:** Neutralizes negative charges on DNA, allowing molecules to aggregate.
• **Phenol:chloroform:isoamyl alcohol:** Removes proteins; isoamyl alcohol reduces foaming and can further inactivate RNases.
• **Isopropanol:** Facilitates DNA precipitation by reducing solubility in water.
• **Ethanol (70%):** Washes away residual salts and solvents.