The sample may be stored at -80☌ for later use, or keep on ice for immediate homogenisation.Place the tissue in a round-bottom microfuge tube or Eppendorf tube and immerse in liquid nitrogen to “snap freeze”.Dissect the tissue of interest with clean tools.Note: For this process we recommend keeping the tissue cool throughout the process to avoid degradation by proteases. Denature the lysate by heating the lysate at 95☌ for 5 minutes.Aspirate the supernatant and transfer to a fresh tube kept on ice, and discard the pellet.Gently remove the lysate from the centrifuge and place on ice.leukocytes need a very light centrifugation). For this step you should adjust the centrifugation force and time depending on the cell type - as a guideline use 20 minutes at 12,000 rpm, but this should be determined by the person carrying out the procedure (e.g. In a cool microcentrifuge, centrifuge the lysate.Maintain constant agitation for 30 minutes at 4☌.
Scrape any adherent cells off the dish using a cold plastic cell scraper before gently transferring the cell suspension into a pre-cooled microfuge tube.(1 ml per 10 7 cells/100mm dish or 150cm 2 flask, 0.5ml per 5x10 6 cells/60mm dish or 75cm 2 flask) Remove the PBS and add ice-cold lysis buffer.Place the cell culture dish on ice and wash the cells with ice-cold Phosphate Buffer Saline (PBS).Check that the confluency of the cells is to expectation by viewing under a microscope.
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