Instructions
The approximate ratio of bases to dye molecules can be determined by measuring the absorbance of the labeled aRNA at 260 nm and at the maximum absorbance wavelength for the dye.

1. Determine the appropriate dilution for spectrophotometer readings.

   Dilute labeled aRNA in 10 mM Tris-HCL, 1 mM EDTA (TE). Mix well and measure A₂₆₀ in a UV-Vis spectrophotometer.

   To obtain an accurate reading at both 260 nm and at the maximum absorbance wavelength for the dye, the aRNA dilution should result in an A₂₆₀ between 0.1 and 1.0. If the A₂₆₀ is below 0.1, reduce the dilution factor and check the A₂₆₀ again.

2. Measure the aRNA absorbance at 260 nm and at the absorbance max for the dye.

   Blank the instrument with the TE used for making dilutions. Measure the absorbance of each sample at 260 nm (A₂₆₀) and also at the maximum absorbance wavelength for the dye used in the coupling reaction (A_dye)

   Dye*:	-- Choose a dye -- Fluorescein Rhodamine Cy3 Cy5 Alexa Fluor 546 Alexa Fluor 555 Alexa Fluor 660 Alexa Fluor 647 Oyster 556 Oyster 645 Oyster 656 Other
   *Please choose "Other" if you would like to use extinction coefficient values and/or maximum absorbance values different from those listed in the table below.

   
   
   

Notes About Calculation

Table 1. Reference Values for Spectrophotometry Calculations.


	Absorbance maximum	Extinction coefficient*
Fluorescein	491	66,000
Cy™3	550	150,000
Cy™5	650	250,000
Alexa® Fluor 546	546	104,000
Alexa® Fluor 555	555	150,000
Alexa® Fluor 660	663	132,000
Alexa® Fluor 647	650	239,000
Oyster 556	556	155,000
Oyster 645	645	250,000
Oyster 656	656	220,000
* Extinction coefficient at Absorbance Maximum in cm-1M-1

 

		Adye		9010 cm-1M-1
#dye molecules per 1000 nt	=		x		x	1000
		A260		extinction coefficientdye

 

• A_dye is the absorbance of the labeled nucleic acid at the dye's maximum absorbance wavelength.
• Extinction coefficient_dye is the extinction coefficient of the dye at the maximum absorbance wavelength.