After spin-vac, resuspend DNA in approximate 5.0 ul of 0.1M TEA pH7.0
The Ellman's reagent reacts with thiols to produce a colored product that absorbs light at 412 nm. The purpose of this test is to verify that we have reactive thiol DNA and to determine the concentration of this DNA.
TO MAKE 0.4% ELLMAN'S REAGENT:
Dissolve 4mg Ellman's reagent [5,5'-dithiobis (2-nitrobenzoic acid)] in 1.0 ml 0.1M TEA pH8.0
OUTLINE:
0.6 ul thiol-DNA
0.4 ul Ellman's
+ 59.0 ul 0.1M TEA pH8.0
60.0 ul à 100 X dilution of thiol DNA
+ 540.0 ul 0.1M TEA pH8.0
600.0 ul à 1000 X dilution of thiol DNA
PROCEDURE:
--- under "Instruments" go to "Lamps", click the "ON" for both circles.
--- under "Methods" go to "Setup Analysis"
a) "Data type" = "Absorbance"
b) "Use wavelengths": type in 260 nm and 412 nm
c) "Display spectrum" = 200nm - 600nm
--- put 59.6 ul 0.1M TEA pH8.0 in the UV-vis cuvet
--- click on the "Blank" button to take a blank measurment
--- click on the "Sample" button
--- to name a sample, simply click in the space of the "name" cell of the data table.