It is well known that SDS and CTAB is a good method for removing protein in DNA extraction, but i am doing extraction of dna of pseudomonas bacteria and i didn't see any good results, although i don't have access to phenol, can anyone have suggestion and help me in dna extraction?
Kais Khudhair al Hadrawi
Prepare Bacterial Culture:Start with a pure bacterial culture grown overnight in an appropriate medium. Harvest Bacteria:Pellet bacterial cells by centrifugation at a low speed (e.g., 3000 × g for 10 minutes) to collect cells. Discard the supernatant carefully. Cell Lysis:Resuspend the bacterial pellet in an appropriate lysis buffer (e.g., TE buffer) containing proteinase K. Optionally, add RNase A to degrade RNA. Incubate the mixture at an appropriate temperature (e.g., 37°C) for a specified time (typically 30-60 minutes) to lyse the cells and release DNA. Extraction of DNA:Add an equal volume of PCI solution to the lysed cells. Mix thoroughly by vortexing and then centrifuge at maximum speed (e.g., 12,000 × g) for 5-10 minutes. Transfer the upper aqueous phase containing DNA to a new tube, avoiding the interface and organic phase. Purification:Add an equal volume of chloroform to the extracted DNA solution. Mix gently by inversion and centrifuge again. Transfer the upper aqueous phase to a new tube. Precipitation of DNA:Add an equal volume of isopropanol to the DNA solution. Mix gently and incubate at room temperature or in a -20°C freezer for 30 minutes to overnight to precipitate DNA. Centrifuge at maximum speed (e.g., 12,000 × g) for 10-15 minutes to pellet DNA. Wash and Dry DNA:Carefully remove the supernatant without disturbing the DNA pellet. Wash the DNA pellet with 70% ethanol to remove residual salts and contaminants. Centrifuge briefly and carefully remove the ethanol. DNA Resuspension:Air-dry the DNA pellet or briefly centrifuge to remove excess ethanol. Resuspend the DNA pellet in TE buffer or nuclease-free water by gentle pipetting or vortexing. Incubate at a suitable temperature to ensure complete dissolution of DNA. DNA Quantification and Storage:Measure the concentration and purity of extracted DNA using a spectrophotometer or fluorometer. Store the extracted DNA at -20°C or -80°C for long-term
DNA extraction from bacteria typically involves several steps to break down the bacterial cell wall and membrane, release the DNA, and remove contaminants. Here's a basic protocol for DNA extraction from bacteria:
Materials Needed:
- Bacterial culture
- Lysis buffer (e.g., Tris-EDTA (TE) buffer, Tris-HCl buffer, or commercial DNA extraction kits)
- Proteinase K
- RNase A (optional)
- Phenol-chloroform-isoamyl alcohol (PCI) solution
- Chloroform
- Isopropanol
- Ethanol (70% and 100%)
- TE buffer or nuclease-free water
Protocol:
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Chithan C Kandaswami
Greetings! You could try the following: Simple boiling. Employing chelex-100. Ultra pure water. Enzymic digestion: lysozyme. Heat and sodium hydroxide. Good luck!
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