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<div class=Section1>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Here is the general method that we use in
Tom's lab:</span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=3 color=black
face="Times New Roman"><span style='font-size:12.0pt;color:black'>To Clean AUC
Cells:</span></font></p>
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>1.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Remove
housing plugs and red plug gaskets.</span></font></p>
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>2.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Remove
the solution from each side of the centerpiece using a pipette and gel tips.</span></font></p>
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>3.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Loosen
the screw ring from the cell housing using the torque stand.</span></font></p>
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>4.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Manually
finish unscrewing the screw ring and remove the underlying screw ring gasket.</span></font></p>
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>5.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Turn
cell housing upside down and push out the window holders and centerpiece gently
using a kim-wipe.</span></font></p>
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>6.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Remove
the windows, window liners, and window gaskets from the window holders.</span></font></p>
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>7.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Wipe
the window liners, window gaskets, and window holders with kim-wipes. If
any are excessively wet, rinse with water/5% contrad/water/ethanol and dry.</span></font></p>
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>8.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Wash
the centerpieces and windows:</span></font></p>
<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>a.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Rinse
with water.</span></font></p>
<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>b.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Rinse
with 5-10% Contrad70 or RBS and either rub manually or use a Q-tip to remove
all protein residues from the windows and centerpieces.</span></font></p>
<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>c.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Rinse
again with dionized water to remove all Contrad.</span></font></p>
<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>d.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Rinse
with ethanol.</span></font></p>
<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>e.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Dry
with Nitrogen.</span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=3 color=black
face="Times New Roman"><span style='font-size:12.0pt;color:black'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=3 color=black
face="Times New Roman"><span style='font-size:12.0pt;color:black'>When working
with RNA do all of the above followed by (Carrie May's Method):</span></font></p>
<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>f.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Soak
windows and centerpieces in RNAseZAP (Ambion cat# 9780.9782) for 2 mins</span></font></p>
<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>g.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Rinse
with DEPC treated water for 2 mins</span></font></p>
<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>h.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Rinse
with ethanol for 2 mins</span></font></p>
<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><font size=3
color=black face=Times><span style='font-size:12.0pt;font-family:Times;
color:black'>i.<font size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font color=black><span style='color:black'>Dry of
nitrogen or let air dry on bench</span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Tahoma><span
style='font-size:10.0pt;font-family:Tahoma'>-----Original Message-----<br>
<b><span style='font-weight:bold'>From:</span></b> rasmb-admin@server1.bbri.org
[mailto:rasmb-admin@server1.bbri.org] <b><span style='font-weight:bold'>On
Behalf Of </span></b>Allen H Pekar<br>
<b><span style='font-weight:bold'>Sent:</span></b> Monday, February 21, 2005
10:25 AM<br>
<b><span style='font-weight:bold'>To:</span></b> rasmb@server1.bbri.org<br>
<b><span style='font-weight:bold'>Subject:</span></b> [RASMB] Procedures for
Cleaning Ultracentrifuge Cells</span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'><br>
</span></font><font size=2 face=sans-serif><span style='font-size:10.0pt;
font-family:sans-serif'>RASMB Members: Tom Laue's e-mail of Feb. 14, 2005
states that he has seen "surface effects (most frequently surface binding
and aggregation)" . He also has seen "differences from window
to window and centerpiece to centerpiece (of the same kind), suggesting that
cleanliness may be a problem". I contacted Tom about recommended
cleaning procedures for centrifuge cells and he will be sending me the methods
used in his lab. Tom also suggested that I ask other RASMB members for
their input. If anyone has a cleaning procedure which they think helps
minimize differences between cells, I would be very interested. Thanks, Allen
Pekar (Eli Lilly and Company, Indianapolis, Indiana) </span></font></p>
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