Merlin maxiprep help?
(by silvergrin from gmail.com)
Tue Jul 17 21:01:26 EST 2007
Thanks for your response!
That's interesting that you use 1-1-1. We used to use a kit and I had
noticed that resuspension-lysis-neutralization ratios for that was
1-1-1.4. And we actually have been using 0.7 V isopropanol, so, in
between your method and the posted one XD
However, this was working before, at these concentrations...
But it's something to try! I'm going to try a couple of midipreps
tonight to see if I can get one of them working again. :P
As to the glucose, I'd love to know! I wondered about it but don't
know why it's there...
On 7/17/07, DK <dk from no.email.thankstospam.net> wrote:
> In article <mailman.301.1184695643.11350.methods from net.bio.net>, Adie <silvergrin from gmail.com> wrote:
> >Hello! My sincerest apologies if you get this twice; I could really
> >use some help here and I didn't see my first email show up...
> >I found this list by looking for Merlin Maxiprep protocol
> >info. It was posted a *long* time ago (1994) but you can still find
> >the protocol here:
> >I've been put in charge of making the Merlin solutions. When we first
> >started using the assay, a couple different people had made them.
> >Everything worked pretty well. We ran low on Merlin I, II, and IV, so
> >I made up a more recently, and, horrors, the prep stopped working. D:
> > The problem is that DNA is not precipitating during the spin-down
> >after adding isopropanol to the filtered lysate.
> >I think the problem must lie with either buffer I or buffer II; we are
> >still using the old stock of buffer III, from when the assay was
> >I've already compared notes with the person who made up buffer I the
> >first time (seems to be the same), re-made buffer I in case I got
> >something horribly screwed up, and also tried a new solution of buffer
> >II. Still no crude DNA pellet at the end of the spin. Does anyone
> >know what the problem could be?
> >From your description, the trouble is with standard alkaline lysis
> protocol, not anything specific to the downstream "Merlin" steps.
> No certain clue here but the protocol in the URL you provided
> looks very unusual to me in that it uses 2X volume of SDS/NaOH
> solution vs normal 1X of the neutralization solution (E.g., usually
> it is 1+1+1 but is 1+2+1 in your protocol). I would suspect that
> this may result in insufficient precipitation of SDS and may
> even not be enough to neutralize NaOH. Both factors, I think,
> make for a less efficient DNA precipitation with alcohols,
> particularly when used with only 0.6V (I am more accustomed to
> a 0.8V) of isopropanol.
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