After spin-vac, resuspend DNA in approximate 5.0 ul of 0.1M TEA pH7.0

 

ELLMAN TEST

 

            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 100 X dilution of thiol DNA

      +   540.0 ul 0.1M TEA pH8.0

            600.0 ul 1000 X dilution of thiol DNA 

 

 

PROCEDURE:

  1. Turn on lamps and let them warm-up for 10-15 min.

--- under "Instruments" go to "Lamps", click the "ON" for both circles.

  1. Set up display

--- 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

  1. Take a blank

--- put 59.6 ul 0.1M TEA pH8.0 in the UV-vis cuvet

--- click on the "Blank" button to take a blank measurment

  1. Add 0.4 ul Ellman's, take another reading (blank 2)

--- click on the "Sample" button

--- to name a sample, simply click in the space of the "name" cell of the data table.

  1. Add 540 ul 0.1M TEA pH8.0, take another reading (blank 3)
  2. empty and clean UV-vis cuvet; combine first three "ingredients" under outline; let react 10 min
  3. Take an absorbance measurement (100 X measuement); add 540 ul 0.1M TEA pH8.0, take an absorbance measurement (1000 X measurement).
  4. Repeat steps 6 and 7 for each sample of thiol DNA
  5. Save your data