Separation Of cDNAmRna Hybrids From Singlestranded cDNAs BY hydroxyapatite Hap Chromatography

1. Carry out preparation of an HAP column: add 1g of HAP (DNA Grade Bio-Gel, BioRad, Cat.# 130-0520) in 0.1 M phosphate buffer (PB), pH 6.8 for 5 ml of slurry and mix well. Heat in boiling water for 5 min. Place a 5-ml plastic syringe closed at the bottom in a water bath to equilibrate at 60°. Place some sterile glass wool at the bottom of the syringe and slowly add 0.5 to 1 ml of HAP slurry to the bottom-closed column and allow it to set for 5 min prior to opening the column. Wash the column twice with 5 volumes of 0.1 M PB (60°).

2. Dilute the hybridized sample 10-fold in prewarmed (60°C) 0.1 M PB containing 0.15 M NaCl. Gently load the sample onto the prepared HAP column with the bottom closed. Gently loosen the mixture in the column with a needle (avoid bubbles) and allow it to set for 10 min.

3. Open the column from the bottom and collect the effluent containing the single-strand cDNAs. Wash the column twice with 0.5 ml of 0.1 M PB containing 0.15 M NaCl and collect the effluent. The cDNA/mRNA hybrids in the column are then eluted with 0.5 M PB.

4. Pool the effluent together and load onto a fresh HAP column as in step 3 and step 4 to obtain the maximum of any cDNA/mRNA hybrids that potentially remain in the effluent.

5. Pool the effluent together and dialyze against 500 ml dd.H2O overnight to remove the PB salts.

6. Add 0.15 volume of 3 M sodium acetate buffer (pH 5.2) and 2.5 volumes of chilled 100% ethanol to the dialyzed sample and place at -80°C for 30 min.

7. Centrifuge at 12,000 x g for 5 min and briefly rinse the cDNA pellet with 1 ml of 70% cold ethanol. Dry the pellet under vacuum for 15 min and dissolve the cDNAs in 20 to 25 ml TE buffer. Take 2 ml of the sample to measure the concentration of cDNA. Store the sample at -20°C until use.

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