<div dir="ltr">Hi
<span style="font-family:"Times New Roman",serif;font-size:13.3333px">Qiaohui</span><div><span style="font-family:"Times New Roman",serif;font-size:13.3333px">I would agree with the 7-8% stock concentration for UA. Even at that concentration, it would take a while to dissolve in methanol. Additionally, I would filter it with a 0.22 micron filter, make aliquots and then freeze in LN2. Dont know if LN2 was overkill but i have never seen cloudy precipitation in my FS mixes and i was using 3% OS and 0.5% UA along with some TA and glutaraldehyde. </span></div><div><span style="font-family:"Times New Roman",serif;font-size:13.3333px"><br></span></div><div><span style="font-family:"Times New Roman",serif;font-size:13.3333px">Hope this helps!</span></div><div><span style="font-family:"Times New Roman",serif;font-size:13.3333px"><br></span></div><div><font face="Times New Roman, serif"><span style="font-size:13.3333px">Best</span></font></div><div><br></div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Thu, Dec 19, 2019 at 11:31 AM Christopher Peddie <<a href="mailto:Christopher.Peddie@crick.ac.uk">Christopher.Peddie@crick.ac.uk</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">
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Sorry, I should have been more specific: 20% in methanol.<br>
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<div id="gmail-m_-7348354367130906389divRplyFwdMsg" dir="ltr"><font style="font-size:11pt" face="Calibri, sans-serif" color="#000000"><b>From:</b> Gibson, Kimberley <<a href="mailto:kimberley.gibson@yale.edu" target="_blank">kimberley.gibson@yale.edu</a>><br>
<b>Sent:</b> 19 December 2019 15:08<br>
<b>To:</b> Christopher Peddie <<a href="mailto:Christopher.Peddie@crick.ac.uk" target="_blank">Christopher.Peddie@crick.ac.uk</a>>; Qiaohui Yang <<a href="mailto:qiaohui.yang@ymail.com" target="_blank">qiaohui.yang@ymail.com</a>><br>
<b>Cc:</b> <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a> <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>
<b>Subject:</b> Re: [3dem] Precipitation observed after making freeze substitution cocktail containing 5% water</font>
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It's not possible to make a stock solution of 20% Uranyl Acetate. Even making a 10% UA stock is not possible. At best, the solubility of UA is around 7-8 % in water. It's best to make a 5% stock solution, dissolved in water, for example, and add the quantity
of 5%UA to the acetone. The precipitation of UA is likely due to the acetone as UA is not really soluble in acetone. </div>
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Best,</div>
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<div style="font-family:Tahoma;font-size:13px">Kimberley Gibson</div>
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Research Associate, Charles Sindelar Lab<br>
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Dept. Molecular Biophysics and Biochemistry<br>
Yale University<br>
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<div id="gmail-m_-7348354367130906389x_divRplyFwdMsg" dir="ltr"><font style="font-size:11pt" face="Calibri, sans-serif" color="#000000"><b>From:</b> 3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu" target="_blank">3dem-bounces@ncmir.ucsd.edu</a>> on behalf of Christopher Peddie <<a href="mailto:Christopher.Peddie@crick.ac.uk" target="_blank">Christopher.Peddie@crick.ac.uk</a>><br>
<b>Sent:</b> Thursday, December 19, 2019 03:56<br>
<b>To:</b> Qiaohui Yang <<a href="mailto:qiaohui.yang@ymail.com" target="_blank">qiaohui.yang@ymail.com</a>><br>
<b>Cc:</b> <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a> <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>
<b>Subject:</b> Re: [3dem] Precipitation observed after making freeze substitution cocktail containing 5% water</font>
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<span style="font-size:9pt;font-family:"Trebuchet MS",sans-serif">Hi Qiaohui,</span></p>
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<span style="font-size:9pt;font-family:"Trebuchet MS",sans-serif"> </span></p>
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<span style="font-size:9pt;font-family:"Trebuchet MS",sans-serif">Mix the osmium, water and acetone and cool to -20oC. Then add the uranyl acetate from a stock e.g. 20%. Cooling the mixture in my experience stops the uranyl acetate from precipitating out.</span></p>
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<span style="font-size:9pt;font-family:"Trebuchet MS",sans-serif"> </span></p>
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<span style="font-size:9pt;font-family:"Trebuchet MS",sans-serif">Best,</span></p>
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<span style="font-size:9pt;font-family:"Trebuchet MS",sans-serif">Chris</span></p>
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<span style="font-size:9pt;font-family:"Trebuchet MS",sans-serif"> </span></p>
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<span style="font-size:9pt;font-family:"Trebuchet MS",sans-serif"> </span></p>
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<b><span style="font-size:11pt;font-family:Calibri,sans-serif" lang="EN-US">From:</span></b><span style="font-size:11pt;font-family:Calibri,sans-serif" lang="EN-US"> 3dem [mailto:<a href="mailto:3dem-bounces@ncmir.ucsd.edu" target="_blank">3dem-bounces@ncmir.ucsd.edu</a>]
<b>On Behalf Of </b>Qiaohui Yang<br>
<b>Sent:</b> 19 December 2019 08:39<br>
<b>To:</b> <br>
<b>Subject:</b> [3dem] Precipitation observed after making freeze substitution cocktail containing 5% water</span></p>
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<span style="font-size:10pt">Hi everyone!</span></p>
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<p style="margin:0cm 0cm 0.0001pt;font-size:12pt;font-family:"Times New Roman",serif">
<span style="font-size:10pt">We recently tried to make freeze substitution cocktail consisting of 1% osmium tetroxide, 0.1% uranyl acetate and 5% water but observed that the mixture turned slightly cloudy within 1-2 minutes. We tried to see if the precipitation
would occur in the absence of osmium tetroxide, and we observed some cloudiness too after mixing uranyl acetate solution in water with pure acetone.</span></p>
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<p style="margin:0px 0cm;font-size:12pt;font-family:"Times New Roman",serif">
<span style="font-size:10pt">Just wondering if anyone has encountered similar problem and if so, would appreciate some tips on how to overcome this problem.</span></p>
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<span style="font-size:10pt">Thank you!</span></p>
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<span style="font-size:10pt"> </span></p>
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<span style="font-size:10pt">Best regards,</span></p>
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<span style="font-size:10pt">Qiaohui</span></p>
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<span style="font-size:10pt"> </span></p>
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</blockquote></div><br clear="all"><div><br></div>-- <br><div dir="ltr" class="gmail_signature"><div dir="ltr"><div><div dir="ltr"><div>Amar Parvate<br></div><div>Saphire Lab</div><div>LJI<br></div></div></div></div></div>