My cells are MCF-7 and MCF-10.
I want extract DNA and RNA from these cell using Trizol.
Do I add Trizol directly into the flask after removing the media? What amount of Trizol is needed if I grow my cells in a 75-t flask?
If I want to do DNA and RNA extractions, do I need to centrifuge before adding chloroform?
Can I use ethanol not treated with DEPC in the RNA wash step?
Can I isolate DNA from the interphase and the phenol-cloloroform phase after separate homogenization?
centrifuge after adding chloroform and
its better to use 70% ethonol from DEPC water
"Do I add Trizol directly into the flask after removing the media? What amount of Trizol is needed if I grow my cells in a 75-t flask?" - You can discard the medium of your cells, wash with 1% PBS, discard the PBS and add trizol directly to the flask. I use about 4-5mL of Trizol to a 80-90% confluent T-75 flask, but you should optimize according to your cells and confluence.
"If I want to do DNA and RNA extractions, do I need to centrifuge before adding chloroform?" - Usually I add to the sample with the trizol (in eppendorfs) the chloroform and then centrifuge.
"Can I use ethanol not treated with DEPC in the RNA wash step?" - Yes, it is important to use during all the process of RNA extraction DEPC treated water to avoid degradation of RNA.
3-4ml Trizol should be fine, but that depends on your cell type. Add (0.2ml/ml of Trizol) chloroform to the lysate and then centifuge. Use DEPC-treated water and a good quality ethanol (Merck's should be fine).
Make sure all of the aqueous phase is gone. Add 100%EtOH and centrifuge again to obtain DNA pellet.
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