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