I wonder if anyone knows a protocol for extracting DNA from fat layer of aspirated bone marrow after centrifugation and layer separations. It would be my pleasure to receive any suggestions.
Here is a protocol for extracting DNA from the fat layer of aspirated bone marrow after centrifugation and layer separations:
Materials:
- Aspirated bone marrow sample with fat layer
- Sterile phosphate-buffered saline (PBS)
- Red blood cell lysis buffer
- Proteinase K
- Phenol:chloroform:isoamyl alcohol (25:24:1)
- Chloroform
- Isopropanol
- 70% ethanol
- TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0)
Protocol:
1. Remove the fat layer from the aspirated bone marrow sample after centrifugation and layer separations using a sterile pipette.
2. Rinse the fat layer with sterile PBS to remove any remaining blood cells.
3. Add red blood cell lysis buffer to the fat layer to lyse any remaining red blood cells. Incubate at room temperature for 5 minutes.
4. Add proteinase K to the fat layer and mix gently. Incubate at 55°C for 1-2 hours to digest proteins and release DNA.
5. Add an equal volume of phenol:chloroform:isoamyl alcohol to the sample and mix well. Centrifuge at 12,000 rpm for 10 minutes.
6. Transfer the aqueous phase to a new tube and add an equal volume of chloroform. Mix well and centrifuge at 12,000 rpm for 10 minutes.
7. Transfer the aqueous phase to a new tube and add 0.6 volumes of isopropanol. Mix well and incubate at -20°C for 30 minutes to precipitate the DNA.
8. Centrifuge at 12,000 rpm for 10 minutes and carefully remove the supernatant.
9. Wash the DNA pellet with 70% ethanol and air-dry for 10-15 minutes.
10. Resuspend the DNA pellet in TE buffer and store at -20°C until use.
Note: It is important to maintain sterile conditions throughout the procedure to prevent contamination of the DNA sample. Additionally, it is recommended to quantify the DNA using a spectrophotometer or fluorometer before downstream applications.
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