Deprotection of IDT thiol modified DNA and Ellman’s test
- Resuspend the crude DNA from IDT in 50mM phosphate buffer pH 8.4. Add 10 uL per each OD260nm unit from the IDT spec sheet.
- Make a 200mM solution of DTT in 50mM pH 8.4 phosphate buffer (15.4 mg in 500uL of buffer)
- Mix equal parts (v/v) of DTT and crude DNA solutions and allow to react for 30 minutes. (total volume should not exceed 200uL)
- Purify through reverse phase HPLC following the instructions on the HPLC
- Speed vac the solutions (~ 8hrs)
o Twist the valve in the hose line from the pump to the cold trap to release the vacuum.
o Remove and replace the cold trap with a fresh one (located under the hood) then reseal the trap and twist the valve back into its original position (with the handle pointing along the hose line)
o Turn on the vacuum pump and the speed vac unit. (the cold trap should be on and it should never be turned off)
o Place samples in centrifuge rack in opposite positions, close speed vac top and press “Manual Run”. Hold down the lid until a pressure reading appears in the gauge next to the unit to ensure a seal.
o Let run until samples have been dried, and should appear as white powder or translucent film)
- Ellman’s test
o Resuspend the speed-vac-ed DNA in 5uL of 100mM TEA pH 7.0 buffer
o The extinction coefficient of the Ellman’s reagent is 13600 M-1cm-1
o Create a 0.4% Ellman’s Reagent solution (4mg Ellman’s reagent [5,5’-dithiobis (2-nitrobenzoic acid)] in 1.0 mL 100 mM TEA pH 8.0 buffer
o Turn on UV-vis lamps and allow them to warm up for 10-15 minutes
§ Instruments ą Lamps ą click “on” for both lamps
o set up the displays
§ Methods ą Setup Analysis
Š “Data Type” = “Absorbance”
Š “Use Wavelengths” type in 260 nm (for the DNA) and 412 nm (for the thiol groups)
Š “Display Spectrum” = 200nm – 600nm
o Blank solutions
§ Put 59.6 uL of 100mM TEA pH 8.0 buffer in micro UV-vis cuvet and click “Blank” button to take a new blank measurement
§ Add 0.4 uL of the Ellman’s Reagent solution and gently mix. Click “Sample” and label this spectrum “100xBlank”
§ Add 540 uL of 100mM TEA pH 8.0 buffer and take another Sample reading. This should be labeled as “1000xBlank”
o DNA readings
§ Empty and clean cuvet with water and ethanol. Dry the inside and out of the cuvet thoroughly. Combine 59.0 uL 100mM TEA pH 8.0 buffer, 0.4 uL Ellman’s solution, and 0.6 uL thiol-DNA solution. Mix gently and allow to react for 10 minutes.
§ After the 10 minutes, take an absorbance measurement (100 x measurement). Then add 540 uL 100mM TEA pH 8.0 buffer. Mix gently and take another absorbance measurement (1000 x measurement)
§ Repeat the last two steps for each thiol DNA sample and save your data.
© Corn Group 2006