Linear acrylamide (was --Re: glycogen as a carrier)

Paul N Hengen pnh at cutterjohn.ncifcrf.gov
Tue Apr 23 11:11:03 EST 1996


(Dave Johnston) (daj at nhm.ac.uk) wrote:

: I then repeated with 500, 250, 125,63, 31, 16, 8, 4, 2, 1 ng. From this, I 
: recogned that I was recovering down to 4ug without loss.

This is great stuff! But, I'm pretty sure you meant 4 nanograms, right?
Did you try various sizes of DNA at any time?

: I now use only 5-15 ul of the LPA soln and often don't bother with the 
: salt either. It seems to work well for cleanup of ABI TaqFS cycle 
: sequencing reactions although we may be getting a spurious set of peaks 
: which we are still investigating.

I suppose the LPA was prepared by the Gaillard.Strauss1990 method?

@article{Gaillard.Strauss1990,
author = "C. Gaillard
     and F. Strauss",
title = "Ethanol precipitation of {DNA} with linear
polyacrylamide as carrier",
journal = "Nucleic Acids Res.",
volume = "18",
number = "2",
pages = "378",
year = "1990"}

| Linear polyacrylamide is made by polymerising a 5% acrylamide
| solution in 40mM Tris, 20mM Na acetate, 1mM EDTA pH7.8 together with
| 1/100 vol of 10% ammonium persulphate and 1/1000 vol. TEMED. When the
| solution becomes viscous (about 15-30mins) ppt polymer with 2.5 volumes
| EtOH, centrifuge and redissolve pellet in 20 vol. water O/N. Use 10ul
| (25ug) of this 0.25% LPA soln per EtOH pptation.

In that paper, the figure 1 shows that less than 20 bp DNA is lost by this
method, which is good if you want to remove primers, linker DNA, or
unincorporated nucleotides, but not good if that's the size you want to
recover. On the other hand, if you use glycogen as carrier, an 8 bp DNA can be
recovered (looks about 50% recovery on the autoradiogram by my keen eyeballs).
Unfortunately, a gel shift shows the glycogen interferes with protein-DNA
interactions, while the LPA did not (figure 2).  Their recovery was on the
scale of 20 picograms of linear DNA.

--
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