My understanding is that glucose is used in the resuspension solution to prevent cells from bursting by maintaining the appropriate osmotic pressure. Why do we even bother doing this? The cells are going to be lysed with the addition of the SDS+NaOH, and we have EDTA in the resuspension solution to chelate Mg, thus inactivating nucleases.


This is a common misconception: in fact, in the alkaline lysis method for plasmid isolation, the glucose does not act as an osmotic stabiliser.

Glucose is present in the resuspension buffer at 50 mM. This concentration is not high enough for osmotic protection of spheroplasts formed by lysozyme treatment: from memory, 0.5 M glucose is typically used for stabilisation. So why is the glucose included?

The original method for plasmid preparation by alkaline lysis was published by Birnboim and Doly in 1979. They explain that the method relies upon achieving a pH of between 12.0 and 12.5 after addition of NaOH so that linear dsDNA (sheared chromosomal DNA) is denatured but closed-circular DNA (plasmid) is not. They state in the paper that:

further pH control is obtained by including glucose as a pH buffer.

According to this site glucose has a pKₐ of 12.3, which aligns nicely with the claim that it helps to keep the pH within the desired range.

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    $\begingroup$ This is an interesting bit of information (I really like the explanations of this mechanisms). Thanks for finding it. $\endgroup$ – Chris Jul 5 '17 at 7:55
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    $\begingroup$ @Chris I actually knew the answer, having been around when the Birnboim & Doly paper was published. This technique revolutionised what was possible with plasmid-based cloning at the time and I read it very carefully. At the time of writing (05-07-2017) the paper has 12,877 citations, but most people who use the technique (in kits) won't even know of its existence. Sic transit gloria mundi $\endgroup$ – Alan Boyd Jul 5 '17 at 8:20
  • $\begingroup$ The kit problem (as I usually call it with our students) is exactly the reason why I love the information bits. They help in trouble shooting and it is good to know how a method works anyway. Classical biochemistry with finding out exact mechanisms. I thought I would know the lysis method and the steps pretty well, but there is always something to learn. As it turns out, this paper is already in my literature database, I will re-read it sometime later. $\endgroup$ – Chris Jul 5 '17 at 8:25
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    $\begingroup$ Really great answer, but the second link is broken for me. $\endgroup$ – canadianer Jul 5 '17 at 16:22
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    $\begingroup$ @canadianer fixed, I hope. $\endgroup$ – Alan Boyd Jul 5 '17 at 17:10

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