Inquiry about DNA recovery by silica binding

John Richard Seavitt jrseavit at artsci.wustl.edu
Mon Oct 20 11:02:30 EST 1997


On Mon, 20 Oct 1997, Wilson wrote:

>  After adding the silica to solution which contain dissolved agarose,
>  DNA and guanidium thiocyanate I tap the solution to mix them.
>  Then should I incubate the mixture in ice or at room temp?

Most protocols I've seen suggest continuing to incubate at 55 degrees
centigrade (i.e., the temperature you melt the agaraose at, so as to keep
it melted).  The binding reaction isn't particularly reversible in the
presence of the choatropic salt, however, and I currently use a RT
incubation.  Take home message: it don't matter.

>  Should I use 50% ethanol with 100mM NaCl, Tris-HCl and EDTA or only 70%
>  ethanol as the washing solution?

All the solutions I've seen are tris-buffered and do have edta and salt,
along with varying proportions of ethanol.  I personally use 50mm salt,
10mM Tris, pH7.5, 2.5 mm edta, and 50% EtOH.  I'm sure the commerical,
proprietary solutions vary somewhat from this, and I'm sure the small
differences don't make much difference.  I've not seen it with just EtOH.

>  Finally,  which incubation temperature can elute effectively?

All the protocols I've seen call for 2-5 min at 55 degrees C.

>  One more question is about the safety of guanidium thiocyanate...
>  guanidium thiocyanate can release very toxic gas in acidic 
>  solution. Is it possible that toxic gas released in the procedure
>  (e.g solution III) of miniprep?

Though I was not aware of this little detail, I'll point out that solution
three of alkalai lysis neutralizes an already basic solution (NaOH in the
lysis buffer).  Since dna is susceptible to acid hydrolysis, you'd better
not be ending up with an acid solution (I've not checked, myself).  I'd
worry more about the large gamish of salts in that solution messing up the
binding reaction, however; why not precipitate with isopropanol, wash with
EtOH, reususpend, and the silica-purify (killing both birds...).  You
should know that the pH does matter in another context; agarose gel
running buffers are often basic (ph >8) and this does inhibit dna binding
to the beads; we give our gel slices a quick rinse with Tris pH7 befrore
proceeding.

John Seavitt





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