I am growing cells in 96 well plates and then essentially performing a western blot in the plate. This involves (among other things):
- Fixing (4% paraformaldehyde/10 min)
- Permeabilising (PBS+0.1% Triton X-100/5 min)
- Washing and antibody incubations (PBS+0.1% Tween-20)
After imaging the plates, I now want to extract DNA, perform a PCR and send it off for sequencing (NGS).
Ideally I'd like a simple protocol to liberate enough (crude) DNA from my cells so that I can amplify small (
I think you can easily exclude step one, and use PCR buffer with DTT.
Otherwise it is possible to add "Prep and go" buffer (from Thermo) instead of PBS to the cells and amplify 1-2mcl after incubation.
Thanks for the tip Andrey. I'm currently looking at Proteinase K, and a few other similar enzymes. I'd rather liberate the DNA in the well, rather than in the PCR reaction, so that I don't have to worry about scraping enough fixed cells off the plate. The Prep-n-Go buffer looks interesting, however it's pretty expensive. If I have no luck with PrK then I'll give it a try. Cheers.
UPDATE: The Proteinase K method we put together worked beautifully. It managed to liberate plenty of DNA for a robust PCR reaction, even for samples with very little input (
Hello Alasdair,
I wanted to extract DNA from very low number of cells (10,000 cells). These cells have been fixed and permeablised for intracellular staining and then flow sorted. I wanted to know more details about your method. Would you be able to provide me a more detailed protocol of the same.
I have to do PCR on theses samples and submit them for sequencing.
Thanks
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