INTRODUCTION
DNA STAT-120 kit includes two solutions for the efficient isolation of high molecular weight DNA. No chloroform, phenol or any poisonous material is used in this method. Does not require centrifugation to isolate DNA. DNA can be isolated in less than 1 hour from samples of human, animal, plant, yeast and bacterial origin and particularly well suited for the simultaneous processing of multiple samples. Both solutions 1 and 2 have an extended shelf life.

Method: All procedures are performed at room temperature. A quantity of 10⁶-10⁸ nucleated cells in suspension should be completely dissolved per 1 ml of solution 1 with the volume in which the cells are suspended making up less than 25% of the total. For example, take 10⁶ cells suspended in 100 ul PBS and dissolve them by adding 300 ul solution 1 and gently inverting the solution several times.

Other starting materials: Erythrocytes should be removed from whole blood by hypnotic lysis. The sample is then centrifuged, the pelleted white blood cells resuspended in PBS or equivalent solution, and then dissolved as above in solution 1. Alternatively, buffy coat of whole blood can be diluted and then used as starting material. Solution 1 can be poured directly on a tissue culture plate to dissolve cells grown in mono layer. Tissue samples should be homogenized and resuspended in solution 1 (10 mg/ml).

Precipitation and purification of genomic DNA: Crude DNA is precipitated by adding 2.5 volumes of absolute ethanol and mixing for 1 minute by gentle inversion. The long white strands of DNA tend to accrete into a single large loose mass which settles quickly to the bottom of the tube. The liquid is decanted and the DNA washed twice with a generous volume of a wash solution made up of 1 vol. 0.3 M sodium acetate/3 vol. absolute ethanol. The excess liquid is removed and the specimen is redissolved in 1 vol. of solution 2. The DNA is then reprecipitated by adding 2.5 vol. of a precipitating solution made up of 1 vol. 2 M sodium acetate/3 vol. absolute ethanol and mixing 1 min. by gentle inversion. The precipitated DNA is washed twice with the gentle inversion. The precipitated DNA is washed twice with the wash solution made up of 1 vol. 0.3 M sodium acetate/3 vol. absolute ethanol, and redissolved in water or TE. If the redissolved DNA is too dilute, add 2.5 vol. of the precipitating solution mentioned above, and proceed accordingly, redissolving in a smaller volume of water or TE at the end.

For PCR applications: Heparin lithium, included in solution 2 is a potent inhibitor of Taq polymerase, but not of most restriction enzymes. This reagent must be removed by enzyme heparinase prior to PCR amplification. 1 ug of DNA can be incubated with 2.5 U of heparinase II (Sigma) for 2 hours at room temperature in a buffer containing 5 mM Tris, pH 7.5, and 1 mM CaCl. For amplification, the PCR cocktail is added directly to the heparinase treated genomic DNA.

Items required but not supplied:
1. Washing solution (1 vol. 0.3 M sodium acetate/3 vol. absolute ethanol).
2. Precipitating solution (1 vol. 2 M sodium acetate/3 vol. absolute ethanol).
These items are available in our Gold pack/ Cat. No. 4510.