[3dem] Single-sided blotting in Vitrobot?

[Bill Tivol]

On Dec 10, 2008, at 1:41 PM, Fred Sigworth wrote:

> Has anyone had success in using the Vitrobot to freeze a specimen  
> that needs blotting only on one side?  On the one hand we have  
> specimens with ultra-thin carbon over a holey film.  The vitrobot  
> quite reliably breaks the films (perhaps due to dry filter-paper  
> fibers contacting them?).  On the other hand, we have streptavidin  
> crystals spanning holes, where the crystals are formed on a lipid  
> monolayer which presents a dry, hydrophobic surface on one side.  It  
> would be great if we could use the Vitrobot to freeze these things.


Dear Fred,
	Try blotting at an edge of the grid.  Set the Vitrobot so it won't  
blot, then reach through the hole used for sample loading with a piece  
of blot paper on a tweezer and touch the edge of the grid.  Hold the  
paper against the edge for the appropriate time, which will likely be  
longer than you think, then plunge.  The grid should have a gradient  
of ice thickness with one part of the grid having ice that's too  
thick, one where it's too thin, and, one hopes, a part where the ice  
is just right.  This is easier to accomplish in the Vitrobot than with  
a gravity plunger, even when one puts the gravity plunger in a cold  
room.  At least until you have worked out blotting times, this will be  
a numbers game, so plan on making a lot of grids and scanning them to  
see which ones have worked.  Good luck.
Yours,
Bill Tivol, PhD
EM Scientist
Ultrafast EM Facility
Noyes Laboratory, MC 127-72
California Institute of Technology
Pasadena CA 91125
(626) 395-8833
tivol at caltech.edu