Phage DNA prep.-Expected Yield?

Duncan Clark Duncan at genesys.demon.co.uk
Wed Apr 20 07:06:41 EST 1994


In article:  <9404200012.AA0060 at bmmb01.umesci.maine.edu>
  rr at BMMB01.UMESCI.MAINE.EDU (Robert Roxby) writes:
> 
> Just out of curiosity, what kind of yield do people end up with in large-scale 
> lambda DNA isolations?  Theoretically there is a mg of DNA in 1L of culture 
> having a titer of 2x10E10.  (Never mind that it seems difficult to get more 
> than 1x10E10).  I can concentrate the phage almost quantitatively with PEG, but
> after 2 CsCl gradients, step and equilibrium, followed by formamide 
> extraction,  I only end up with about 20% of the theoretical amount of DNA. 
> Not that this is bad as things go (try purifying an enzyme sometime), but the
> books, such as CPMB, seem to imply that you will get more than that.  Also, if
> you only end up the 400 ng/liter of culture, I don't see how the commercial
> outfits can sell you 10 ug for $60, although I am sure 
> it isn't CsCl purified.  Experiences or enlightenment appreciated.
> 
> 
> Robert Roxby                           rr at bmmb01.umesci.maine.edu
> Biochemistry, Microbiology             rroxby at maine.maine.edu
>    & Molecular Biology                 72053,1712
> University of Maine
> 

Ah well you see we in the business cheat my using decent lysogens. I can get
(CsCl purified!) approx 10mg per litre of methylated lambda from a cI857sam7
lysogen in W1485 but only 1/2mg per litre from similar dam-dcm-JM110 lysogen.
I'd love to know why the dam-dcm- lysogen gives poorer yields. 

Duncan
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Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
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