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</o:shapelayout></xml><![endif]--></head><body lang=EN-US link=blue vlink=purple><div class=WordSection1><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D'>Dear Peter,<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D'>Thank you for your paper. It is useful to know that it is possible to measure species of this size using direct boundary approaches, also useful and interesting that a high dilution is necessary to avoid electrostatic effects – this is probably because of the charge on the nucleic acid? <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D'>I wonder if at this dilution, 0.005%wt/v, if there would be sufficient signal for a 10MDa species to use the absorbance optics. I would assume so since nucleic acids absorb strongly at 260nm, but my assumptions oft go awry. Perhaps another reason to go down the CsCl road.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D'>Best regards,<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D'>John Sumida<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D'>Molecular Analysis Facility<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D'>University of Washington<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D'><o:p> </o:p></span></p><div><div style='border:none;border-top:solid #E1E1E1 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b><span style='font-size:11.0pt;font-family:"Calibri",sans-serif'>From:</span></b><span style='font-size:11.0pt;font-family:"Calibri",sans-serif'> Prevelige, Peter Edward, Jr [mailto:prevelig@uab.edu] <br><b>Sent:</b> Thursday, June 06, 2019 5:58 PM<br><b>To:</b> John Sumida <jpsumida@uw.edu><br><b>Cc:</b> Borries Demeler <demeler@gmail.com>; RASMB <rasmb@list.rasmb.org><br><b>Subject:</b> Re: [RASMB] AUC measurements in CsCl?<o:p></o:p></span></p></div></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal style='margin-bottom:12.0pt'>Maybe worth noting that it was important in the M&S experiment that the DNA was sheared. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1307605/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1307605/</a><o:p></o:p></p><div id=AppleMailSignature><p class=MsoNormal>Sent from my iPad<o:p></o:p></p></div><div><p class=MsoNormal style='margin-bottom:12.0pt'><br>On Jun 6, 2019, at 7:44 PM, John Sumida <<a href="mailto:jpsumida@uw.edu">jpsumida@uw.edu</a>> wrote:<o:p></o:p></p></div><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><div><p class=MsoNormal>Dear Borries,<br><br>Thank you for your response which is 100% on the mark with regards to the<br>expected deliverables. <br><br>While the work being proposed is not an exact repeat of the Meselson & Stahl<br>experiment (one of my favorites as well) it is along the same vein. The<br>work is attempting to use the methods outlined in the Flory Vinograd work<br>(which I've attached) which uses bromodeoxyuridine as a mass label (an<br>interesting idea ) instead of N15; and yes, my collaborators are seeking to<br>know the relative quantity of the species detected and the sedimentation<br>value is less important. <br><br>Interesting also that in the Messelson and Stahl experiment they used whole<br>cell lysate, this was not significant to me when I read this paper back in<br>the day, but now this seems particularly useful?<br><br>I do not have the 3mm cells here, do you think this experiment is still<br>doable in a standard two channel SV cell?<br><br>Just so I am clear on the reason to use CsCl in this particular case is the<br>ability to detect small amounts of DNA, the large masses involved, which<br>would require a long slow speed spin, and avoid complications from<br>heterogeneity that may be existant at a given buoyant density and which may<br>be difficult to interpret?<br><br>Thanks so much for this information, I am eager to get started.<br><br>Best regards,<br><br>John Sumida<br>Molecular Analysis Facility<br>University of Washington<br><br>In many spheres of human endeavor, from science to business to education to<br>economic policy, good decisions depend on good measurement.<br>Ben Bernanke<br><br><br><br><br><br>-----Original Message-----<br>From: Borries Demeler [<a href="mailto:demeler@gmail.com">mailto:demeler@gmail.com</a>] <br>Sent: Thursday, June 06, 2019 4:36 PM<br>To: John Sumida <<a href="mailto:jpsumida@uw.edu">jpsumida@uw.edu</a>><br>Cc: RASMB <<a href="mailto:rasmb@list.rasmb.org">rasmb@list.rasmb.org</a>><br>Subject: Re: [RASMB] AUC measurements in CsCl?<br><br>Hi John,<br>Accurate molar mass measurement of any solute by SV or SE methods requires<br>an accurate vbar. This varies depending on ionic strength, ie.<br>hydration - so, do you know that? And if so, how? Cesium will bind to the<br>DNA changing its vbar (it is a very dense molecule). SE will also be<br>challenging since equilibrium will take forever to be achieved at lower<br>speed, and the steep gradient you are likely going to see at the bottom of<br>the cell will be distorted by refractive effects and not give you a lot of<br>points. Moreover it would be nearly impossible to resolve any heterogeneity<br>in the samples from size. So, I wouldn't use SE.<br><br>For SV experiments to additionally get a molar mass you need to be able to<br>measure the diffusion coefficient accurately. Large molecules like this have<br>very little diffusion signal. You would have to run for a very long time at<br>a very slow speed to get sufficient accuracy.<br><br>Everything else requires making assumptions and estimates. So I think your<br>traditional approaches for SV and SE will not be satisfactory.<br><br>It sounds like your collaborators are asking for the classic experiment<br>performed by Meselson & Stahl in 1958 (paper attached, one of my favorites,<br>I make my students read it). The different amounts of incorporated label<br>impart slightly different molar masses that can be separated based on the<br>different densities using buoyant density equilibrium gradients. It is the<br>CsCl gradient that will make the two populations buoyant at different places<br>in the cell. As you can see in the paper, this works quite well, and even<br>better with modern equipment.<br><br>For this to work well in an AUC, I recommend using 3 mm centerpieces.<br>It minimizes the CsCl gradient signal and hence attenuates the refractive<br>effect from the gradient. Use optically pure CsCl so you don't have<br>background at 260 nm where the DNA will absorb. This *should* work, though I<br>haven't tried it. While it will not give you accurate molar masses it should<br>resolve the two populations of labeled and unlabeled DNA. As an added bonus,<br>any heterogeneity from shearing is pretty much masked, since all molecules<br>will go where they have the same density, which differs for labeled and<br>non-labeled molecules. Speeds between<br>40-50 krpm should work well. Let us know how it works out. You won't need a<br>lot of DNA to make this measurement, since all the DNA will migrate to a<br>small spot and concentrate there to make a big peak.<br>Follow the paper to get the CsCl concentration correct. This is important<br>and needs to be matched to the DNA samples so you get the best separation.<br><br>In the end you still don't know absolute molar masses but I don't think you<br>actually need that. I bet your collaborators only care about the relative<br>amount of each population. You could also try this by standard velocity<br>analysis, but there the result will also encounter concentration dependent<br>non-ideality, but may resolve the boundaries from the two different<br>densities. Heterogeneity may be reducing your confidence here.<br>Meselson and Stahl, though an old paper, still seems like the most elegant<br>way to solve this problem. Let us know how it works out.<br><br>Good luck!<br><br>-Borries<br><br>Thu, Jun 06, 2019 at 02:49:44PM -0700, John Sumida wrote:<br><br><o:p></o:p></p><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>Dear RASMB,<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>First I have to thank all those who curate this list server as over <o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>the last<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>10 years as a manager of the lab I run, it has and continues to prove <o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>a hugely valuable resource for me and the people I serve.<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>My question today has to do with the analysis of DNA and it stems from <o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>a user interest in performing an AUC equilibrium experiment in the <o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>presence of CsCl. My experience to date has been with nanomaterials <o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>and proteins so DNA is a new material for me, however I am under the <o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>impression that running AUC samples in presence of CsCl is <o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>problematical because of the gradient CsCl itself will generate.<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>The estimated mass of the species of interest is in the range of 10 <o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>mega Daltons; this is also a size regime that is new to me.<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>The objective of the experiment is to characterize nucleic acids <o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>extracted from cells which will have a label increasing the mass of <o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>the nucleic acid depending on the number of times it has been replicated.<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>Two questions:<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>Is CsCl necessary to determine the size distribution of molecular <o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>species<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>10MDa?<o:p></o:p></p></blockquote></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>Can a CsCl experiment be performed using equilibrium measurements?<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>Thank you for considering my question.<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>Best regards,<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>John Sumida<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>Molecular Analysis Facility<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>University of Washington<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>In many spheres of human endeavor, from science to business to <o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>education to economic policy, good decisions depend on good measurement.<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>Ben Bernanke<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><o:p> </o:p></p></blockquote><p class=MsoNormal><br><br><o:p></o:p></p><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>_______________________________________________<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal>RASMB mailing list<o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><a href="mailto:RASMB@list.rasmb.org">RASMB@list.rasmb.org</a><o:p></o:p></p></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><p class=MsoNormal><a href="http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org">http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org</a><o:p></o:p></p></blockquote><p class=MsoNormal><o:p> </o:p></p></div></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><div><p class=MsoNormal><Flory Vinograd BrdU mtDNA 1973.pdf><o:p></o:p></p></div></blockquote><blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'><div><p class=MsoNormal>_______________________________________________<br>RASMB mailing list<br><a href="mailto:RASMB@list.rasmb.org">RASMB@list.rasmb.org</a><br><a href="http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org">http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org</a><o:p></o:p></p></div></blockquote></div></body></html>