DNA ligation-- Peter's freezing method

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Tue Mar 12 10:43:08 EST 2002


Here's a message from Peter at Harvard detailing some more points of his
freezing/spinning gel purification method that I mentioned in yesterdays
post.  Thanks Peter!

Mike

>
>Hi Mike,
>
>Thanks for your reference to me-- after reading it, there were a few other
>things I thought I should have mentioned: the method works fine with
>either regular or low-melt agarose; if use low melt, you just seem to get
>a smaller agarose pellet.  (I tend to use low melt crystal violet, which
>means that: 1) the DNA is never exposed to UV; and 2) I have the option of
>either using it directly, like has been mentioned, or doing the
>purification on it-- i.e., it increases my options, which I like).  The
>only other thing worth mentioning on this point is that I also tried it
>once with high concentration (2-3%) agarose, and it didn't work well-- I
>think there just isn't enough liquid in the agarose in that case, or the
>density of the agarose prevents it from being removed. (When I tried doing
>it, I found very little liquid supernatant was coming out.)  This doesn't
>surprise me overmuch, since you won't have good luck with Spin-X'ing high
>concentration agarose either, and will probably have to do something which
>solubizes the agarose instead-- like Geneclean or agarase.
>
>Also, if you try something other than liquid nitrogen (if you're curious),
>what you find is that the agarose hasn't completely precipitated out--
>forms more of sort of a slick, shiny viscous mass than a strong pelletable
>precipitant. I learned that one the hard way one time when I couldn't find
>any liquid nitrogen around; the problem is that, once you've done the dry
>ice/ethanol rather than the liquid nitrogen, then doing the liquid
>nitrogen on it won't work (you still won't get a nice tight pellet), so
>it's really better just to use the liquid nitrogen to start with.
>Probably something about the way that the agarose precipitates out.
>
>Take care,
>
>Peter
>

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

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