can someone assist me with a protocol to extract genomic DNA from cyanobacteria using phenol-chloroform?
Dear Stephen
- I have never personally done this but I imagine the answer is almost certainly yes:
- Once your cells are lysed and DNA etc. has been released into an aqueous environment the way that phenol and chloroform remove impurities like lipids carbohydrates and especially protein is essentially the same for all DNA regardless of source
- The issue tends to be lysing the cells to release DNA in the first place:
- This is where protocols for extracting DNA from different organisms tend to differ and extra substances are required as part of the initial lysis, e.g. lysozyme is used to break open the cells walls in gram positive bacteria for DNA/RNA extraction
- Something similar might apply to sourcing DNA from cyanobacteria and providing you can lyse the cells efficiently in the first place purifying DNA by phenol chloroform will work
Hi dear Musasa
This paper can be useful for your study.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC106599/
Dear Stephen
- I have never personally done this but I imagine the answer is almost certainly yes:
- Once your cells are lysed and DNA etc. has been released into an aqueous environment the way that phenol and chloroform remove impurities like lipids carbohydrates and especially protein is essentially the same for all DNA regardless of source
- The issue tends to be lysing the cells to release DNA in the first place:
- This is where protocols for extracting DNA from different organisms tend to differ and extra substances are required as part of the initial lysis, e.g. lysozyme is used to break open the cells walls in gram positive bacteria for DNA/RNA extraction
- Something similar might apply to sourcing DNA from cyanobacteria and providing you can lyse the cells efficiently in the first place purifying DNA by phenol chloroform will work
1. A certain amount of cells equivalent to 1 OD730 units of culture were harvested by centrifuging at 12, 000rpm for 1 min;
2. Adding silica sand.
3. Then, 100ul saturated phenol (saturated phenol is in the lower layer, upper level is protection liquid) and 100ul Chloroform: isoamyl alcohol (v/v 24:1) were added. And cells were disrupted with silica sand and those organic regents by vigorous vortex (5 min);
4. After centrifugation at 12, 000rpm for 5min, you can collect and transfer the supernatant into a new tube . Then you can add Chloroform: isoamyl alcohol (v/v 24:1), whose volume was similar to the supernatant. After mixing with chloroform and isoamyl alcohol thoroughly and centrifugation, the supernatant was collected.
5. After the addition of 1/5V(Volume) NaCl and 2~3V ethanol (100%) , you can mix them thoroughly and then set them at -20℃ for 30min at least.
6. After centrifugation at 12, 000rpm for 10min, you can remove the supernatant. Then you can add 500ul ethanol (70%) to wash the DNA.
7. After centrifugation at 12, 000rpm for 5min, you can remove the supernatant. Then the precipitates were collected and dried at room temperature for 3~5min.
8. You can add 30~50ul ddH2O or TE to dissolve the DNA.
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