Hello everyone,
Can anyone please let me know how can I extract DNA of Cyanobacteria (Gram Positive bacteria) in on site?
Extracting DNA from Cyanobacteria, which are gram-positive bacteria, can be a bit challenging, but it's possible to do it on-site with the right equipment and protocols. Here's a general outline of the steps involved in extracting DNA from Cyanobacteria:
Cell harvesting: Cyanobacteria can be harvested from various sources such as soil, water, or biological samples. For on-site extraction, you can use a sterile loop or swab to collect the cells from the surface of a culture or sample.
Cell lysis: The next step is to break open the cell membrane and release the DNA. You can use a lysis buffer, such as Tris-EDTA, to break down the cell membrane and release the DNA. You can also add a detergent such as SDS to help break down the membrane.
DNA release: After lysis, you can add a DNA release agent such as proteinase K to break down any proteins that may be binding to the DNA. This will help to release the DNA from the cellular debris.
DNA purification: The DNA released from the cells will be in a mixture of cellular debris and other contaminants. You can use a purification method such as phenol-chloroform extraction or ethanol precipitation to separate the DNA from the contaminants.
DNA concentration: After purification, you can use a DNA concentration buffer, such as ethanol or isopropanol, to precipitate the DNA and concentrate it.
DNA analysis: Finally, you can analyze the DNA using various techniques such as PCR, restriction digestion, or sequencing.
Here's a simple protocol for on-site DNA extraction from Cyanobacteria:
Collect the Cyanobacteria samples using a sterile loop or swab.
Add 500 μL of lysis buffer (Tris-EDTA, pH 8.0) and 100 μL of 20% SDS to the samples.
Vortex the mixture for 10-15 minutes to break down the cell membrane.
Add 50 μL of proteinase K (10 mg/mL) to the mixture and incubate it for 30 minutes at 50°C.
Add 500 μL of phenol-chloroform to the mixture and vortex it for 10-15 minutes.
Centrifuge the mixture at 10,000 x g for 10 minutes to separate the DNA from the contaminants.
Collect the DNA pellet and wash it with 70% ethanol.
Centrifuge the DNA pellet again at 10,000 x g for 10 minutes to remove the ethanol.
Dry the DNA pellet for a few minutes and then re-suspend it in 100 μL of TE buffer (Tris-EDTA, pH 8.0).
Analyze the DNA using PCR, restriction digestion, or sequencing.
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