DNA transfer

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Fri Jan 13 14:27:48 EST 1995


Jeff Krammer wrote:

> 	The S & S guy just told us about the new turbo blot, which will alleged-
> ly allow one to transfer DNA to a nylon membrane in ~90 minutes.  Has any one
> tried turbo blotting, and would you care to share any advice/opinions on said
> blotter.  

It's basically a plastic tray designed to set up a downward transfer.  It 
looks convenient for their particular sized gel.  On the other hand, you
could probably rig a downward transfer from stuff you find laying around
the lab just as well, and then you could blot any size gel you want.

Like any transfer, the time to transfer will depend on the thickness
of the gel, its percentage of agarose, how large the fragments are, and 
whether or not you chopped them up with acid/alkali or UV treatment.

The downward tranfer probably goes faster than upwards transfer because
you get more flow under gravity than under capillary action.  Until
seeing evidence to the contrary, I'd assume the transfer is complete
when the same amount of fluid goes through the gel/cm^2, regardless
of how it was moved through.  There is the claim that upward transfer
is slowed because the weight on the gel causes it to collapse, but
I assume that by the time the buffer leaves the gel, the DNA has already
left with it.  

They put a comparison in their flyer showing that the Turbo blot was
10x more sensitive than a standard upward SSC over night blot onto
Nytran.  But their hybridization to the standard blot was actually less
sensitive than an EtBr stain.  You all need to be aware of this common
advertising ploy of making the alternative look bad so that
the new product looks good in a side by side comparison.


> For that matter, any advice on turbo blotting vs slot blotting 
> (vacuum) vs. electroblotting would be welcome.  

As far as I know, electroblotting is the only thing that works out of
polyacrylamide.  If there are now other methods, I'd like to hear 
about them.  The vacuum and pressure gizmos can also be fast.  They
have the advantage that they speed up the pretreatments of the gels
as well as the actual transfer.  But I like to run larger gels for
the greater resolution and the extra slots for more controls.  These
invariably got torn when I tried to get them into the vacuum gizmo.
For putting DNA straight on the filter (no gel) it's hard to beat
a vacuum slot blotter.

The old capillary transfer, like a good laxative, works over night while you
sleep and never fails you.  If you start 3 experiments in the morning 
instead of one, you'll lose your desire for everything to be fast, fast,
fast.

> Something that has always
> wondered me is, why is the sensitivity of slot blotting sooo much better than
> electroblotting?  ...

Slot blotting is nearly 100% efficient.  Nearly all the DNA is on the filter
and in a form accessible to hybridization.  For other blotting methods
you get losses for a variety of reasons:
1) DNA left in gel -  Shouldn't be a problem except with large DNA that
                      you didn't break up.  Can be evaluated by staining
                      gel with EtBr after transfer.
2) DNA passes through filter - Mainly small fragments, and mainly a 
                               problem with nitrocellulose.  You may
                               be able to push the DNA through the 
                               charged nylon filters with electro
                               transfer; I don't know.
3) DNA renatures during blot - that's why the alkaline transfers work
                               a little better than SSC.
4) DNA embeds in filter where probe can't get to it - Evaluate by measuring
                     the amount of DNA bound both by having radiolabelled
                     it and hybridizing to it.  The charged membranes work
                     a little better because they trap the DNA at the surface.
5) DNA washes off filter - mainly only a problem with overzealous washing 
                           between repeat hybridizations
6) Hybridization obscured by physical interaction of DNA with membrane -  Some
                           of the sequence has to be tied up in the membrane
                           interaction.  Usually this would only be a factor
                           for very small fragments, but you can inhibit
                           hybridization signal with overzealous UV treatment
                           of the membrane.

In my hands, most methods only differ by a factor of 2, with the best to
worst range being less than a factor of 10.  So 100 x loss of efficiency
would suggest to me that something isn't right in the procedure.  But I
haven't any personal experience with electroblotting.

Steve Hardies, Dept. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu





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