Spec vs Flourimeter for [DNA] ?

Leonard N. Bloksberg bloksber at pilot.msu.edu
Fri Dec 3 08:24:00 EST 1993


In Article <754872157snz at genesys.demon.co.uk> "Duncan at genesys.demon.co.uk (Duncan Clark)" says:
> Hi Netters,
> 
> In response to the query raised about my earlier post I was thinking along 
> the lines mentioned by Jim ie just because you are using CsCl and can't see 
> any RNA does not mean that there isn't any!
> 
> Regarding internal controls, the most reliable I think would be lambda DNA 
> or similar ie something isolated from phage particles. My logic is that the
> somewhat 'harsh' pretreatment (prior to DNA extraction from the particles) of
> phage particles ie DNAse, RNAse, PEG pptation and banding in CsCl, should 
> remove all interfering substances for either spec. or fluorimeter assays.
> 
> Just my fourpenneth.
> 
> Duncan 
> -- 
> -----------------------------------------------------------------------------
> Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
> GeneSys Ltd.                        | Compuserve:  100015.1406 at compuserve.com
> -----------------------------------------------------------------------------
> 
.
In more follow up from my origional post, I RNase digest my plasmid preps
before loading on CsCl, and double banded.  I remove EtBr by extracting with
Butanol.  My practice is to extract until I see no color in the butanol
phase, and then 3x more to be sure.   As for trying Lambda DNA, I 
isolated some wt Lambda for gel standards recently.  Phage were RNase/DNase
digested, PEG pptd, CsCl banded twice, then proteinaseK digested, Phenol 
extracted 2x, Phenol/CHCl3/IsoA extracted 1x, CHCl3/IsoA extracted 1x, and
dialysed to extinction (5x in 1 liter).  My 2 tubes gave 500 and 495 ug/ml
on the spec and 355 / 351 ug/ml on the flourimeter.  I don't know why.
The final note is, I don't think I will ever use this peice of equipment 
again.  It actually gives no better sensativity than the spec.  It requires
2ul of sample between 10ug/ml and 1000ug/ml.  I have 300ul cuvettes, for 
my spec, and 3ul in 300ul of TE, from a 10ug/ml DNA solution will give 
me the same barely readable accuracy on my spec.  Given all the problems 
people have reported with the flourimeter, I say why bother?  If your 
thinking of buying one of these albatrosses, save your money for a good spec.
It's a much more versatile, and reliable instrument.
.
.	Leonard N. Blokserg
.	Bloksber at pilot.msu.edu
.
.



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