Oligo Extinction Coefficient Calculator
(RNA degradation procedure below, scroll...).
This web page was given to us from the Williamson Lab.

This form can be used in two different ways. You can either enter your sequence (lower/upper case, spaces OK) or enter the number of each nucleotide (in addition to the absorbance, pathlength*, sample volume of stock measured, total volume in cuvette*, and total volume of your stock). If you choose to enter the number of each nucleotide, be sure that the sequence textbox is empty (the 'Clear Form' button will ensure this). Also, make sure to choose whether your oligo is DNA (the default) or RNA.

*Defaults have been set for these values, but they can be changed.
Concentrations are based on the sum of the extiction coefficients for the monomers, NOT on nearest neighbor method.


DNA RNA
Name of Oligo:
Sequence:
Absorbance at 260 nm: Pathlength* (cm):
Volume of stock measured (uL): Total volume in cuvette* (uL):
Total stock volume (uL):
Number of As: Number of Cs: Number of Gs: Number of Ts:
Number of Us:   

Total oligo length:
Total oligo mass (Da):
Millimolar extinction coefficient (mM-1 cm-1):
Stock Concentration (uM or pmol uL-1):
Total pmols in stock:
Total mass in stock (ug):
Tm of DNA oligo (°C):
Tm calculation for DNA oligo is based on the following equation: Tm = 81.5 + 0.41(%GC) - 675/length of oligo

RNA concentration Assay:

Purpose: To accurately measure the concentration of an RNA sample. RNA secondary structure induces hyperchromicity in the UV absorption of nucleotides. This procedure hydrolyzes the RNA to nucleotides to rmove the hyperchromicity and allows for repeated measurements to reduce the inccuracy associated with this procedure (mostly pipetting error). This procedure hydrolyzes your RNA so it is probably not worth doing unless you are doing at least a 1ml transcription.

1. Aliquot 2 µ L of your RNA sample into each of three 0.5 ml eppendorf tubes.
2. Aliquot 2 µ L of ddH2O each into three more tubes.
3. Add 8 µ L of 1 M NaOH into each tube.
4. Incubate the tubes at 37 ° C for at least one hour.
5. Add 8 µ L of 1 M HCl to each tube.
6. Add 282 µ L of ddH2O (make sure to invert the tube, or spin it down in the centrifuge quickly to get any condensation from the top of the tube).
7. Using the water tubes as blanks, and the micro quartz cuvettes (300 µ L) , take the absorbance of each solution at 260 nm.
8. Average the three readings and use that value to determine the concentration (make sure to use your specific RNA sequence) using the programs installed on the website (Extinction Coefficient Calculator).

Notes: Depending on how accurately you desire to know your concentration, three readings may not be enough. You should make sure that your RNA is in at least 100 µ L so that your assay doesn’t use up too much RNA.