Hello scientific community, I am looking for a protocol for DNA extraction of protoplast from P. patens.
Raghad Mouhamad
Protoplast suspension from P. patens DNA extraction buffer: 100 mM Tris-HCl (pH 8.0), 50 mM EDTA, 500 mM NaCl, 2% SDS alcohol (25:24:1)Phenol:chloroform alcohol (24:1)Chloroform Isopropanol (cold) Ethanol (70%) RNase A (optional, for RNA-free DNA) Microcentrifuge tubes and pipettes Prepare Protoplast Suspension:Ensure protoplasts are in a clean, isotonic buffer suitable for P. patens, often 0.5 M mannitol with some calcium chloride (CaCl₂). Cell Lysis:Centrifuge the protoplast suspension gently (around 500-800 x g for 5 minutes) to pellet the protoplasts. Carefully remove the supernatant and resuspend the protoplasts in 500 µL DNA extraction buffer. Gently mix by inverting, avoiding vigorous shaking to prevent shearing. Incubate the sample at 55°C for 30 minutes to ensure complete lysis. Phenol-Chloroform Extraction:alcohol (25:24:1) to the lysed sample. Mix gently by inverting. Add an equal volume of phenol:chloroformCentrifuge at 12,000 x g for 10 minutes to separate phases. Carefully transfer the upper aqueous layer to a new tube, avoiding any interphase material. Chloroform Extraction:alcohol (24:1) to further purify the sample. Mix gently. To the collected aqueous layer, add an equal volume of chloroformCentrifuge again at 12,000 x g for 5 minutes and transfer the upper aqueous layer to a new tube. DNA Precipitation:Add 0.6 volumes of cold isopropanol to the aqueous layer to precipitate DNA. Invert gently to mix. Incubate at -20°C for 30 minutes (optional but may improve yield). Centrifuge at 12,000 x g for 10 minutes to pellet the DNA. Wash and Resuspend DNA:Wash the DNA pellet with 1 mL of 70% ethanol. Centrifuge at 12,000 x g for 5 minutes. Carefully remove the ethanol, air-dry the pellet briefly, and then resuspend in 30-50 µL TE buffer or nuclease-free water. (Optional) RNase Treatment:If RNA is present and needs to be removed, add RNase A to the DNA solution and incubate at 37°C for 30 minutes.
For Physcomitrella patens (P. patens) protoplast DNA extraction, you could try the following general protocol tailored for moss protoplasts. This method focuses on gentle lysis to protect the delicate protoplasts and uses a standard extraction buffer and a phenol-chloroform purification approach for DNA isolation.
Protocol for DNA Extraction from Physcomitrella patens Protoplasts
Materials:
Protocol Steps:
Tips:
- Use fresh, sterile reagents and avoid introducing shearing forces to preserve DNA integrity.
- Ensure all steps are carried out gently to protect the integrity of protoplast-derived DNA.
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Abhijit Mitra
Harvest Protoplasts: Centrifuge the protoplast suspension at 1000 g for 5 minutes to pellet the cells and carefully discard the supernatant. Lysis Buffer: Resuspend the pellet in a lysis buffer (e.g., CTAB buffer or SDS-based buffer), and incubate at 65°C for 30 minutes to lyse cells and release DNA. Chloroform Extraction: Add an equal volume of chloroform-isoamyl alcohol (24:1), mix gently, then centrifuge to separate phases. DNA Precipitation: Transfer the aqueous phase to a new tube, add isopropanol, and incubate at -20°C to precipitate DNA. Wash and Resuspend: Wash the DNA pellet with 70% ethanol, air dry, and resuspend in TE buffer or nuclease-free water.
For DNA extraction from protoplasts, you can follow this quick protocol:
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