Hi all,
In the past I have used a lysis buffer for bacterial RNA/DNA extraction that contained Guanidine Hydrochloride, N-lauryl sarcosine and LDS. I used it with spin columns. I can't find the original reference from the literature and I don't remember what the amounts of each component were and the pH. Does anyone know this lysis buffer and can remind me how to make it?
Kind regards,
Martha
Hi Martha,
I haven't got the exact answer you're looking for, but the buffer we use for column-based gel extraction of DNA from agarose is 20mM TrisHCl pH6.5, 5.5M Guanidium Isothiocyanate and I usually acidify it with 50µl of mini prep solution 3 (3M Sodium or Potassium acetate) after the gel block has dissolved in the above at 42˚C. You need the pH to be in the 6s to get the DNA to bind to the silica of the column, whether you're working with gel extracted or bacterial DNA as fundamentally the process is identical once you get to the column stage.
If this is basically an alkaline lysis, the solution B (cell lysis buffer) is a mixture of sodium hydroxide and SDS - 0.2N of the former and 1% of the latter if I remember correctly. LDS is less prone to precipitation in the cold than SDS. After you've popped the cells with this you need to neutralise / acidify with the Potassium acetate solution fairly promptly to avoid damaging the DNA.
Is this along the track of the information you're looking for? Classic alkaline lysis protocols are all available online at places like the Cold Spring Harbor lab protocols list - if you can be a bit more specific about what it is you're trying to do it should be possible to be a bit more helpful - please feel free to come back here and discuss this further.
Best wishes, Robin
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