Paul.xu at 22:28, 18 January 2017

2017-01-18T22:28:37Z

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Preparation of the column material

1 g of carboxymethyl-cellulose (CM-cellulose) is suspended in 50 ml of 0.5 N NaOH and stirred for 30 min. After decantation from the supernatant the material is washed with water until the effluent reaches a pH’of 8. The cellulose derivative is suspended in about 100 ml of water and the pH is adjusted to 3.5 by addition of 0.01 N HCl. After filtration under suction the material is washed three times with 5 ml ethanol/ether 1: 1 (v/v) each, followed by 20 ml of ether and finally dried for 60 min at 40°C. 20 mg of calf thymus DNA dissolved in 10 ml water are added to the prepared CM-cellulose. The resulting suspension is spread over the surface of a Petri dish (about 10 cm in diameter) and dried slowly over a period of 60 h at 40°C during which the drying process is controlled by periodically covering the dish. The resulting DNAcellulose is scraped from the surface of the dish, pulverized, suspended in 50 ml of 0.05 M phosphate buffer at pH 7.0 containing 50% glycerol (v/v) and left for 24 h at room temperature. The material is then washed 10 times with 50 ml of water each and stored at 4°C in a 1 M NaCl solution. If the recommended cellulose is used the resulting column material contains about 15 mg DNA per g of dry CM-cellulose which corresponds to about 2.5 mg per ml bedvolume.

Fractionation of DNA polymerases on DNAcellulose

All column operations are carried out at 3°C and pH 7.6 in buffer A (0.02 M Tris-HCl, 1 mM EDTA, 0.5 mM 1 ,Cdithioerythritol, 10% (vol.) glycerol) containing 6 mg/l of the protease inhibitor phenylmethylsulfonylfluoride. After equilibration of the column with buffer A containing 0.1 M NaCl about 1 mg of protein per ml bedvolume is applied (2 vol./h). The column is then washed with 2-3 vol. of the equilibration buffer, 2-3 vol. of buffer A containing 0.2 M NaCl and the enzyme activity is finally eluted with buffer A containing 0.6 M NaCl at a speed of 4 column volumes per h. The column is regenerated in situ by washing with 1 M NaCl and thereafter may be used for several times without any detectable loss in the binding capacity.

DNA-Cellulose - Revision history

Paul.xu at 22:28, 18 January 2017