[RASMB] AUC measurements in CsCl?

[Prevelige, Peter Edward, Jr]

Hi John

There is a body of literature on this. We have relatively recently published a paper using SedV to size phage chromosomes (at ~45Kbp perhaps somewhat larger than your target). However, you might find some hints and hopefully some useful references in it.  https://www.sciencedirect.com/science/article/pii/S0042682217300673?via%3Dihub

Best,

Peter


Sent from my iPad

On Jun 6, 2019, at 5:56 PM, Lake Paul <lake.n.paul at gmail.com<mailto:lake.n.paul at gmail.com>> wrote:

John,

To answer your first question, CsCl is not needed to determine the MW of your DNA. Just make sure have the appropriate ionic strength in solution. You will be spinning this slowly, e.g. 10000 rpm or lower. One sample at a time and scan as fast as possible.

Equilibrium on such a large particle is going to take literally forever. I would not do an equilibrium experiment on a 10 MDa particle.

The SV experiment should suffice to get a pretty good molecular weight.

Lake


On Thu, Jun 6, 2019, 5:51 PM John Sumida <jpsumida at uw.edu<mailto:jpsumida at uw.edu>> wrote:
Dear RASMB,

First I have to thank all those who curate this list server as over the last 10 years as a manager of the lab I run, it has and continues to prove a hugely valuable resource for me and the people I serve.

My question today has to do with the analysis of DNA and it stems from a user interest in performing an AUC equilibrium experiment in the presence of CsCl.  My experience to date has been with nanomaterials and proteins so DNA is a new material for me, however I am under the impression that running AUC samples in presence of CsCl is problematical because of the gradient CsCl itself will generate.

The estimated mass of the species of interest is in the range of 10 mega Daltons; this is also a size regime that is new to me.

The objective of the experiment is to characterize nucleic acids extracted from cells which will have a label increasing the mass of the nucleic acid depending on the number of times it has been replicated.

Two questions:
Is CsCl necessary to determine the size distribution of molecular species >10MDa?
Can a CsCl experiment be performed using equilibrium measurements?

Thank you for considering my question.

Best regards,

John Sumida
Molecular Analysis Facility
University of Washington

In many spheres of human endeavor, from science to business to education to economic policy, good decisions depend on good measurement.
Ben Bernanke


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