I want to extract the DNA from these above-mentioned cell lines by using the organic solvent method. I don't want to use any isolation kit from any brand.
I'm using a very simple method which give me a nice DNA yield without using any special kits or buffers, I tried with 293T cells , A549 and HAP-1 cell line and always works perfectly for PCR.
1-Collection of cells either by scraping or Trypsinzation
2-Pelleting at 8000g for 10 min , then wash it once with PBS
3- Resuspension of cell pellet in TE buffer with 0.1% Triton x100
4-Boiling at 100C for 5 min
5- Centrifugation at 13000g/10 min
6- Collection of Supernatant (DNA) and keep it at -20C till use.
Wish you all luck
We currently used Triazol/Chloroform method to isolate RNA, but you can isolate DNA too following further steps. See link
https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf
You can try traditional CTAB/phenol-chloroform DNA extraction protocol.
try a phenol-chloroform DNA extraction after a cell homogenezitation (and clearing the lisate)
or you can even use a ion exchange chromatography with clear lysate, it isn´t a very conventional protocol, but it is very efective, and its the principle of all those kit of qiagen, i have tried before and works very well
I'm using a very simple method which give me a nice DNA yield without using any special kits or buffers, I tried with 293T cells , A549 and HAP-1 cell line and always works perfectly for PCR.
1-Collection of cells either by scraping or Trypsinzation
2-Pelleting at 8000g for 10 min , then wash it once with PBS
3- Resuspension of cell pellet in TE buffer with 0.1% Triton x100
4-Boiling at 100C for 5 min
5- Centrifugation at 13000g/10 min
6- Collection of Supernatant (DNA) and keep it at -20C till use.
Wish you all luck
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