I'm using PCI method for genomic DNA extraction from E. coli. I wonder will the concentration of SDS and Proteinase K in my lysis buffer be a consideration for a good yield of DNA?
Thank you for responding my question.
I would say 'no' - within limits - and as long as you use enough. I usually use 0.1% to 1% SDS. Proteinase K is a protein and it will constitute a trivial amount of the protein in the extract and will be removed in the Ph-CHCl3 extraction. The SDS might hang around if you use too much, especially if you ethanol precipitate (though I suspect SDS is quite ethanol-soluble). If you dialyse, it certainly won't be a problem.
I would say 'no' - within limits - and as long as you use enough. I usually use 0.1% to 1% SDS. Proteinase K is a protein and it will constitute a trivial amount of the protein in the extract and will be removed in the Ph-CHCl3 extraction. The SDS might hang around if you use too much, especially if you ethanol precipitate (though I suspect SDS is quite ethanol-soluble). If you dialyse, it certainly won't be a problem.
If you ethanol precipitate, use sodium chloride (0,2 M final) rather than sodium acetate to avoid SDS precipitation.
Andrew, I think the original reference is in the Laboratory Manual by Maniatis et al. 1982.
- Badges
- Science method