Streptomyces DNA isolation: Difference between revisions

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== Streptomyces DNA Isolation ==
== Streptomyces DNA Isolation ==
''From 5 mL of culture''
''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 ===
# 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.
# Add 50 µL 10% SDS and mix thoroughly.
# 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.*
# Add 85 µL 5 M NaCl.
# Add 400 µL phenol:chloroform:isoamyl alcohol (25:24:1). Vortex 30 s. Centrifuge 10 min at ≥8000 rpm.
# Transfer the aqueous (top) phase to a new tube, avoiding the organic layer.
# Add 400 µL chloroform:isoamyl alcohol (24:1). Vortex 30 s. Centrifuge 10 min at ≥8000 rpm.
# Transfer the aqueous (top) phase to a new tube.
# Add 0.5 mL isopropanol. Mix by inversion and incubate 5 min at room temperature.
# Spool out DNA or centrifuge at 10,000 rpm to pellet DNA.
# Wash pellet with 1 mL cold 70% ethanol.
# Decant ethanol and allow DNA to air-dry 30–60 min. 
  *Do not overdry — DNA will be difficult to dissolve.*
# 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.

Latest revision as of 15:32, 4 September 2025

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.
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