Correction factor for spectrophotometrical quantitation of DNA

Dilip Dias MAD2631 at rsgis4.tamu.edu
Thu Apr 4 14:30:12 EST 1996


CECILIA
When you measure DNA quantity based on the 260 it is also better to 
measure the 280 value which indicates the amount of certain proteins 
(proteins with tyrosine and tryptophan).  The 260/280 value is a better 
predicter of the protein contamination. It should be between 1.7 to 1.9 
for DNA in purified form. A lesser value (<1.7) indicates that proteins 
are present and a higher value (>2.0) indicates RNA and dNTP 
contamination.  Also it is better to obtain a value for 320 to correct 
for change in the placement of the cuvette.  Once set for for 0 value 
for the blank, it should be 0 for samples too.  If any changes occured 
in the path of the light when placing the sample 320 value will be 
changed and both 260 and 280 values will be changed by the same 320 
value.  For example if 320 value for blank is 0 and for sample it is 
0.25 then 0.25 will be added to your samples 260 and 280 values thus 
producing an overestimate.  This is a real possibility when dealing with 
micro cuvettes and specially if only 260 value is estimated. Some new 
spetrophotometers are programmed to read all three values and to deduct 
the 320 value from 260 and 280 values before calculating the 260/280 and 
concentration of DNA.
Hope this helps.

Dilip Dias
Dept. of Forest Science
Texas A&M university 





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