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Quantitation of phage DNA - EtBr vs OD260

Quantitation of phage DNA - EtBr vs OD260 - Protocols and Methods Forum

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  #1  
Old 06-09-2005, 08:43 AM
C Coward
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Default Quantitation of phage DNA - EtBr vs OD260



I have prepared some bacteriophage DNA but I'm suspicious of my
quantitation of it. When run on agarose and compared to a dilution of
lambda commercial DNA standard I estimate the concentration to be much
lower (by at least an order of magnitidue) than when I take OD260
readings. The OD260/280 ratio is fine (1.966), and particularly strikingly
when I take OD260/280 readings of a dilution of the lambda standards the
spec readings correleate extremely well with the known concentration of
the lambda DNA.
I'm wondering whether there's any possibility that modifications of the
phage DNA may result in the EtBr not intercalating properly, or
fluorescing differently to 'standard' DNA.

Any comments?

TIA

Chris
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  #2  
Old 06-09-2005, 02:42 PM
Raymond Dalgleish
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Default Quantitation of phage DNA - EtBr vs OD260

C Coward wrote:

Remember that OD260 is measuring everything that absorbs at 260 nm which
includes RNA and degraded DNA etc. The 260/280 ratio is suspiciously
high and tends to idicate that the absorbing material is probably RNA,
rather than DNA. A good DNA prep typically has a 260/280 ration of about
1.8 to 1.85 whereas RNA ratios are closer to 2.00.

I always trust ethidium staining.

Raymond
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  #3  
Old 06-09-2005, 04:40 PM
C Coward
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Default Quantitation of phage DNA - EtBr vs OD260

Raymond Dalgleish <[Only registered users see links. ].ac.uk> writes:



Me too, however I've just had another look at my OD results and for some
of the samples the ratio is better (1/100 dilution of phage DNA gives
OD260=0.113; OD280=0.063; 260/280=1.794). Additionally the lambda DNA that
I used was a commercially bought standard, so it should be free of RNA! My
phage preps were also treated with RNase (I do see RNA contamination of
the purified phage when a portion is run on an agarose gel in SDS/EDTA
loading buffer, but the contaminating band completely disappears following
digestion with RNase). Do free nucleotides absorb at OD260? I really
should know that shouldn't I?

Chris
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  #4  
Old 06-10-2005, 06:38 AM
DK
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Default Quantitation of phage DNA - EtBr vs OD260

In article <d89rdr$748$[Only registered users see links. ].cam.ac.uk>, C Coward <[Only registered users see links. ].cam.ac.uk> wrote:

Yes you should!

As others said, unless DNA is very pure (and that is NOT
the case with any tipical miniprep or similar technique), the
spec reading almost invariably overestimates [DNA]. Go with
the EtBr estimate.

DK


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dna , etbr , od260 , phage , quantitation


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