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<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=771271010-09022005>Dear
Oscar and all,</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=771271010-09022005></SPAN></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=771271010-09022005>Automated plunging is very desirable, especially to get
reproducable results, for students and people with little experience and for
certain special cases (temperature and moist critical samples: liposomes,
microtubules, cells and others). I have no experience with the GATAN plunger and
little with the Vitrobot.</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=771271010-09022005></SPAN></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=771271010-09022005>We
found that especially the lower part of the original vitrobot, holding the
ethane, is poorly designed. The grid has to go through air when going from
ethane to the grid container in the nitrogen, the ethane freezes up quickly, and
the grids are very difficult to place inside their containers, which lie deep in
the bubbling nitrogen. So we build our own device mainly changing the lower
part, adapting it a bit to the design of the old Reichert KF80 plunger. We
build in a small heating device and a special chamber to hold the grid boxes,
which are shielded from air and do not suffer from any bubbling of
nitrogen. Now we can operate our machine a full day without refilling any ethane
(which is usually not necessary...). Our machine has also been proven to be
usefull for freezing cells (single layers). If you want I can send you a picture
of the lower part.</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=771271010-09022005></SPAN></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=771271010-09022005>I have
had no experience with the water layer thickness ranging in thickness over the
grid, but I can imagine that if the blotting time is short, the rubbers on the
upper side get no time to not touch the grid properly or are shielded by the
tweezers. Also the grid might be located a bit too high between the filter
papers, so that only part of the grid is blotted properly. You might wanne
try different blotting paper (???). </SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2></FONT> </DIV>
<DIV><SPAN class=771271010-09022005><FONT face=Arial color=#0000ff size=2>I have
to add that I found that treating your grid right is important for getting
good samples. I have the impression that on all home made holey grids (I never
got around making usable ones myself) and also on commercial grids, there is
always some plastic resin left. So what helped me A LOT is that I put thin
layers of carbon ON BOTH SIDES of the grids I buy. Critical seems to be that
directly before use I glow discharge the grid ON BOTH SIDES. It seems that this
increases the spreading of the sample on the grid. This also when using
proteins that prefer to stick to your grid and are not located in the holes, and
I had very good results using ribosomes and no problem at all in getting them in
the holes. I must say that this might ONLY be advantageous when you put the
sample on BOTH sides of your grid, which I always do...</FONT></SPAN></DIV>
<P><FONT face=Arial size=2>Roman</FONT> </P>
<P><FONT face=Arial size=2>R.I.Koning, Ph.D.</FONT> <BR><FONT face=Arial
size=2>Molecular Cell Biology</FONT> <BR><FONT face=Arial size=2>Leiden
University Medical Center</FONT> <BR><FONT face=Arial size=2>Wassenaarseweg 72,
2333 AL</FONT> <BR><FONT face=Arial size=2>Leiden, The Netherlands</FONT>
<BR><FONT face=Arial size=2>(+31) 71 527 6463 (tel)</FONT> <BR><FONT face=Arial
size=2>(+31) 71 527 6440 (fax)</FONT> </P></BODY></HTML>