I have lung tissue. It is an infected tissue. It was obtained from animal necropsy. The goal is to obtain maximum bacteria and maximum bacterial DNA without using an extraction kit and microfilter. How can this be done?
For extracting bacterial DNA from infected lung tissue without using a kit or microfilter, here is a step-by-step protocol that relies on simple reagents and techniques:
### Materials Needed
- PBS (Phosphate Buffered Saline) or sterile water
- Lysis buffer (e.g., 10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
- Proteinase K (20 mg/mL)
- SDS (Sodium Dodecyl Sulfate) 10%
- RNase A (10 mg/mL, optional)
- Phenol:Chloroform:Isoamyl Alcohol (25:24:1)
- Chloroform
- 70% Ethanol
- 100% Ethanol
- Sterile tubes
- Centrifuge
### Protocol
1. **Homogenize Tissue:**
- Place about 100 mg of the infected lung tissue in a sterile tube.
- Add 1 mL of PBS or sterile water.
- Homogenize the tissue thoroughly using a sterile pestle or homogenizer until no large pieces remain.
2. **Cell Lysis:**
- Transfer the homogenized solution to a fresh tube.
- Add an equal volume of lysis buffer and 10% SDS (final concentration 1%) to help break down cellular membranes.
- Add Proteinase K (final concentration 200 µg/mL).
- Incubate the mixture at 55°C for 1-2 hours, or until the tissue is fully digested.
3. **RNase Treatment (Optional):**
- To remove RNA, add RNase A (final concentration of 100 µg/mL) and incubate for 15 minutes at 37°C.
4. **Phenol:Chloroform Extraction:**
- Add an equal volume of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) to the lysate.
- Vortex briefly to mix, then centrifuge at 10,000 x g for 10 minutes at 4°C.
- Carefully transfer the upper aqueous layer (containing DNA) to a new tube without disturbing the interface.
5. **Chloroform Wash:**
- Add an equal volume of chloroform to the transferred aqueous layer.
- Vortex briefly and centrifuge again at 10,000 x g for 5 minutes.
- Transfer the upper aqueous phase to a new tube.
6. **DNA Precipitation:**
- Add 2 volumes of cold 100% ethanol and 0.1 volumes of 3 M sodium acetate (pH 5.2) to the aqueous phase.
- Mix gently by inverting the tube several times, then place at -20°C for 30 minutes to overnight.
7. **DNA Pellet Washing:**
- Centrifuge at 10,000 x g for 15 minutes to pellet the DNA.
- Wash the pellet with 70% ethanol by adding ethanol, inverting gently, and centrifuging again for 5 minutes.
8. **Dry and Resuspend DNA:**
- Air dry the DNA pellet (do not over-dry).
- Resuspend in an appropriate volume of TE buffer or sterile water (around 50-100 µL).
### Tips for Maximizing Bacterial DNA Yield:
- **Avoid excessive tissue:** Using too much tissue can lead to contamination and difficulties in the purification process.
- **Minimize RNase if bacterial RNA isn’t problematic:** If bacterial RNA might aid downstream applications, you can skip the RNase step.
- **Ensure proper temperature:** Proteinase K works optimally at 55°C.
This method should yield bacterial DNA suitable for downstream applications such as PCR. For higher purity, you may repeat the phenol:chloroform extraction and ethanol precipitation steps if needed.
Materials Needed
- Infected lung tissue (approximately 100 mg)
- Phosphate-buffered saline (PBS) or sterile water
- Lysis buffer (e.g., Tris-HCl with EDTA)
- Sodium dodecyl sulfate (SDS) to a final concentration of 1%
- Proteinase K at a final concentration of 200 µg/mL
- Phenol:Chloroform:Isoamyl alcohol (25:24:1)
- Ethanol (70% and absolute)
- Sterile tubes and pipettes
- Homogenization device (e.g., Bullet Blender https://www.nextadvance.com/bullet-blender-homogenizer/)
Step-by-Step Protocol
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