Kais Khudhair al Hadrawi
Algal cells Lysis buffer (containing Tris-HCl, EDTA, SDS, and sometimes proteinase K) Phenol:chloroform:isoamyl alcohol (25:24:1) Chloroform:isoamyl alcohol (24:1) Ethanol Isopropanol RNase A (optional, if RNA contamination is a concern) TE buffer (Tris-EDTA buffer) Harvest the algae cells by centrifugation and wash them with PBS buffer to remove any contaminants. Resuspend the algal cells in lysis buffer and incubate at an appropriate temperature for cell lysis. This buffer should break open the cells and release the DNA. Optionally, you can add RNase A to the lysis buffer to degrade any RNA that might be present. After cell lysis, add an equal volume of phenol:chloroform:isoamyl alcohol to the lysate. Mix the solution thoroughly by inverting the tube gently. Centrifuge the mixture at high speed to separate the aqueous phase (containing DNA) from the organic phase. Carefully transfer the aqueous phase (top layer) to a new tube, avoiding the interface. Repeat the phenol:chloroform:isoamyl alcohol extraction step to ensure complete removal of contaminants. Precipitate the DNA by adding 2-2.5 volumes of cold ethanol to the aqueous phase. Incubate the mixture at -20°C or -80°C for about 30 minutes to allow DNA precipitation. Centrifuge the mixture at high speed to pellet the DNA. Carefully remove the supernatant and wash the DNA pellet with 70% ethanol to remove any residual salts and contaminants. Air dry the DNA pellet or use a vacuum concentrator to remove the ethanol completely. Resuspend the DNA pellet in TE buffer or nuclease-free water. This will be your total genomic DNA extract. The plasmid DNA, being smaller in size, remains in the supernatant during the ethanol precipitation step. You can perform an additional precipitation step using isopropanol to selectively precipitate the plasmid DNA. Precipitate the plasmid DNA by adding 0.6 volumes of isopropanol to the supernatant. Incubate the mixture at -20°C or -80°C for about 30 minutes. Centrifuge the mixture at high speed to pellet the plasmid DNA. Wash the DNA pellet with 70% ethanol and air dry or vacuum concentrate as before. Resuspend the plasmid DNA pellet in TE buffer or nuclease-free water.
Separating genomic DNA and plasmid DNA from algae can be achieved through a process called DNA extraction. Here's a general guide on how you can do it manually:
Materials Needed:
Procedure:
1. Cell Lysis:
2. DNA Extraction:
3. Plasmid DNA Separation (Optional):
Notes:
- Ensure proper handling of hazardous chemicals and biological materials.
- Maintain sterility during the procedure to avoid contamination.
- Adjust the protocol based on the specific characteristics of your algae species and the intended downstream applications of the extracted DNA.
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Alessandro Paparella
and then proceed with the quiagen kit protocol. For the plasmid Invitrogen gene jet plasmid kit is recommended
if your starting material is so sticky, the reason is the presence of fatty acid in the cell membrane, my personal advice before starting the protocol, is to dry it by washing it in CTAB 4% and then to centifugate at 14.000 rpm.
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Nadia Kushmar
Thank you so much Kais Khudhair al Hadrawi for your complete answer. I really appreciate.
I will do it and share the results here.
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