alkaline lysis - (Aug/08/2005 )

i wish to know why we are using

a) Sodium acetate ph 4.8 * why must use this pH scale?
Alkaline lysis
c) SET (sucrose, EDTA, Tris-Cl)
d) TE buffer

for miniprep DNA plasmid extraction

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a) This solution neutralizes NaOH in the previous lysis step while precipitating the genomic DNA and SDS in an insoluble white, rubbery precipitate.
It works? sorry, I don't know...
c) The sucrose makes the solution dense enough so that only the genomicDNA/SDS pellet do not pellet during centrifugation (my guess, not 100% of this one)
c) and d) Tris is a buffer that maintain pH to value that won't degrade DNA too much. EDTA chelates the divalent cation that are usually needed by DNAses thus protecting DNA from degradation.

HTH

Patrick

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I've always used NaOAc pH 5.2 for my alkaline lysis preps, but I've seen pH 4.8 in different protocols. Not sure what effect the pH actually has, but 5.2 works fine for me.

-Hank

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DNA EtOH precipitates better (ie at a low conc.) at acid pHs as there is less charge on the phosphate backbone. Acid pH help remove proteins as well.

The purpose of the sucrose is to prevent shearing of the DNA during manipulation. It also acts as a buffer at ph12.5.

TE prevents degradation of the DNA in two ways: The Tris stops the DNA solution from becoming acidic due to CO2 absorption and the EDTA chelates Mg require by DNases.

Daniel

DNA sequencing analysis software

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