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bluechampagne
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Topic Started by bluechampagne
on 8/4/2006 14:18 PM   
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I have purified adenoviruses on a caesium chloride gradient and then subjected selected bluish white band to dialysis using a 10mM Tris pH8 2mM MgCl2 5 % sucrose buffer ( 200x viral sample volume) with 3 changes.

However i notice that there is a precipitated fraction....
If this is my virus, what is the best way to resuspend it, and get over this precipitation problem

thanks


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Tracy
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Posted By Tracy
on 8/8/2006 23:35 PM   
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Does your supernatant contain enough of Adenovirus? If so, you might not worry about the precipitate.



yeritzaihc
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Posted By yeritzaihc
on 12/10/2007 18:12 PM   
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Hi.

I am trying to do a virus purification. But I don't have MgCl2 aviable to do the dialysis Buffer . Can I change it for another reagment?



yeritzaihc
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Posted By yeritzaihc
on 12/10/2007 18:13 PM   
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Hi.

I am trying to do a virus purification. But I don't have MgCl2 aviable to do the dialysis Buffer . Can I change it for another reagment?



Tony Rook
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Posted By Tony Rook
on 12/10/2007 21:57 PM   
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Hi veritzaihc -

I'm not sure if there is a substitute for MgCl2 but you may find this reference helpful...

S Zolotukhin, B J Byrne, E Mason, I Zolotukhin, M Potter, K Chesnut, C Summerford, R J Samulski, and N Muzyczka. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Nature Gene Therapy. June 1999, Volume 6, Number 6, Pages 973-985.

Abstract -
Conventional methods for rAAV purification that are based on cesium chloride ultracentrifugation have often produced vector preparations of variable quality and resulted in significant loss of particle infectivity. We report here several novel purification strategies that involve the use of non-ionic iodixanol gradients followed by ion exchange or heparin affinity chromatography by either conventional or HPLC columns. These methods result in more than 50% recovery of rAAV from a crude lysate and routinely produce vector that is more than 99% pure. More importantly, the new purification procedures consistently produce rAAV stocks with particle-to-infectivity ratios of less than 100, which is significantly better than conventional methods. The new protocol increases the overall yield of infectious rAAV by at least 10-fold and allows for the complete purification of rAAV in 1 working day. Several of these methods should also be useful for large-scale production.

Tony Rook



parvoman
Scotland

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Posted By parvoman
on 12/11/2007 18:05 PM   
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bluechampagne said:
I have purified adenoviruses on a caesium chloride gradient and then subjected selected bluish white band to dialysis using a 10mM Tris pH8 2mM MgCl2 5 % sucrose buffer ( 200x viral sample volume) with 3 changes.

However i notice that there is a precipitated fraction....
If this is my virus, what is the best way to resuspend it, and get over this precipitation problem

thanks



As Tracy suggested the precipitate may not be a problem if you can just pellet and dispose of it without loosing your infectious virus.

CsCl has been shown by some labs to lead to a reduction in infectious titre of AAV, but I don't think there is a problem with adenovirus preps. In fact I don't think there really is a problem with AAV either.

There are a lot of Adenovirus purification kits available now that are based on affinity chromatography and although they usually do not allow the same high yields of virus that you can get with CsCl gradients, they are a lot faster and do not require any dialysis (which is always going to lead to a loss of virus).

I personally tend to still favour the CsCl prep over affinity preps because they allow you to select for virus particles that contain DNA, since these are more dense. Affinity methods will give you a lot more empty Ad capsids. (Incidentally, the iodixanol based method that Tony suggested is very good for selecting the DNA-containing particles from an AAV prep in the same way if you use it to create a true gradient rather than the discontinuous "cushion" described in the paper.)

When I have removed my Ad virus from the second round of density centrifugation I just desalt it over a PD-10 column. You collect the fractions coming off of the column and OD spec them (260nm). Voila. No nasty loss of virus during dialysis.

Hope this helps

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Hassanvet
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Posted By Hassanvet
on 12/5/2008 1:51 AM   
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please see this link:

http://www.millipore.com/pressroom/pr3/press_release04072008

Billerica, Massachusetts—April 7, 2008—Millipore Corporation, a Life Science leader providing technologies, tools and services for bioscience research and biopharmaceutical manufacturing, today announced the availability of its two Fast-Trap Virus Purification and Concentration Kits, one for adenovirus purification and one for lentivirus purification. Purifying virus after propagation in host cells is a critical step towards using the viruses as vectors for genetic modification of target cells. The Fast-Trap Virus Purification and Concentration Kits contain all the components to accommodate the entire virus purification workflow.



kata.f
New Zealand

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Posted By kata.f
on 10/6/2011 15:14 PM   
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Do you know a kit or method similar to Fast-Trap which works for Rotavirus?



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