DNA Purification - Removal of γ-32P-ATP from Oligonucleotides


Jeff Lawrence

1. Prepare Whatman DE-52 according to manufacturers specifications. Equilibrate at store with 0.02% Sodium azide.

2. Plug a 1 mL (blue) pipet tip with Siliconized glass wool. Add 500 µL of 50:50 DE-52 slurry. Pack into a 250 µL column by gently forcing the fluid through with a P-1000 pipetter.

3. Rinse column twice with 1 mL TE, forcing through with a P-1000.

4. Add 200 µL TE to Kinased Oligo. Apply the solution to the column and gently force though with the P-1000.

5. Elute nucleotides by washing 4 times with 1 mL 0.2 M NaCL in TE. Monitor counts removed. Most residual ATP should be removed after the first 2 rinses.

6. Elute the labeled oligonucleotide with 500 µL 1 M NaCl in TE. Repeat 3 times, collecting each rinse separately. The first rinse should contain most of the oligonucleotide.

7. Up to 10% of the counts may remain on the column.

Last Update: Thursday June 19 2014
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Eric Kofoid eckofoid at ucdavis.edu