DEPROTECTING DNA

Thiol-DNA is received from the UW Biotech Center dried and protected with a trityl protecting group, so it needs to be resuspended and deprotected.

1. Resuspend dried oligo with 250 uL of 100 mM pH 7 TEAA (triethylammonium acetate). Place this in a 1.5 mL Eppedorf tube and label 'CRUDE' with DSN # and date.

2. Remove a 50 uL aliquot of crude DNA from eppendorf. Put in another eppendorf tube.

3. Add 7.5 uL 1M AgNO3. Let react 30 min. This removes the trityl group.

4. Add 10.0 uL 1M DTT (dithiothreitol). Let react 5 min.

This precipitates AgNO3. Do not vortex this; the precipitate may stick to the sides of the vial.

5. Centrifuge for 2 minutes at 14,000 rpm. Transfer supernatant to HPLC vial.

This solution contains deprotected thiol-DNA and excess DTT. It should be about 50 uL.

6. Wash precipitate with 50 uL 0.1 M TEAA pH 7, centrifuge 2 min., add supernatant to HPLC vial.

NOTES:

-- Use deprotected, purified thiol-DNA within 3 weeks.

-- DTT: MW = 154.25 g/mol

0.0077g makes 50 uL of 1M DTT. Dissolve in water. Does not dissolve well. May need to add more water to dissolve better. OK to use even if solution is a bit cloudy.

8-99 KF

10-99 BPN, ed.

Copyright 1999 Corn Group