ANDC DU/Biology Protocols/seperation Single and double strand

Principle
At low ionic strength, double stranded DNA binds to hydroxylapatite (calcium phosphate gel) while single stranded DNA or RNA gets washed out of the column. This differential behaviour is exploited for separation of double stranded DNA from single stranded DNA as well as from RNA.

Materials and Reagents
1. Boiling water bath 2. Small chromatographic column or disposable 3 ml plastic syringe may be used as a column 3. Eppendorf centrifuge tubes 4. Hydroxylapatite slurry: Suspend hydroxylapatite powder (Bio-Gel HTP) in 0.01 M sodium phosphate buffer (pH 6.8) 5. 0.01 M sodium phosphate buffer (pH 6.8) 6. 0.12 M sodium phosphate buffer (pH 6.8) 7. 0.4 M sodium phosphate buffer (pH 6.8) 8. 2-Butanol 9. Total DNA (Precipitate DNA from the preparation with 2 volumes of chilled ethanol. Air dry the pellet and dissolve in appropriate volume of 0.01 M sodium phosphate buffer (pH 6.8) so as to get a solution containing 100-200 μg DNA/ml buffer. Boil the solution in a boiling water bath for 5 min and allow to cool slowly at 25ºC).

Procedure
1. Plug the base of column or syringe with glass wool and pour enough slurry of hydroxylapatite into it to obtain a column with a packed volume of about 1.0 ml. 2. Wash the column with several volumes of 0.01 M sodium phosphate buffer (pH 6.8). 3. Load the sample containing the nucleic acid onto the column and continue to pass about another 3 ml of this buffer through the column. 4. Drain the excess of buffer and add one bed volume (1ml) of 0.12 M sodium phosphate buffer (pH 6.8) preheated to 60°C and collect 0.5 ml fractions each. 5. Now add one bed volume (1ml) of 0.4 M sodium phosphate buffer (pH 6.8) preheated to 60ºC. 6. Collect 0.5 ml fractions. Repeat Step 5 two times more. Let the elute cool down to room temperature. 7. For extracting double stranded DNA, add an equal volume of 2-butanol to each fraction containing the nucleic acid as indicated by Absorbance measurement at wavelength 260nm recorded with the help of spectrophotometer. Mix by vortexing and centrifuge at 12,000×g for 20 sec in an Eppendorf centrifuge tube at room temperature. 8. Discard the upper organic phase and repeat Step 7. 9. Again discard the organic phase and recover DNA from aqueous phase by precipitation with 2 volumes of ethanol at 0ºC.