Principles of Selected Molecular Biology Techniques

Since all steps are carried out on the plate, obviating the need for centrifugation, mixing, or transferring of samples, this procedure is adaptable to automation (Rl). Another reason why proteases should be removed is that the DNA 

Residual phenol can inhibit Taq polymerase. Hence, a final extraction with chloroform—isoamyl alcohol (49 1) should be performed after phenolization to remove any trace quantities of phenol remaining in the aqueous phase (R3). 

RNA can be selectively extracted into the aqueous phase by adjusting the pH 

Since hematin inhibits Taq polymerase, it is absolutely essential to eliminate red blood cell contamination. Selective lysis of red blood cells can be accomplished with a buffer mixture consisting of 155 mM ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM EDTA adjusted to pH 7.4. Alternatively, the cytoplasmic membrane of all cells can be dissolved with a buffer mixture containing the non-ionic detergent Triton-X 100, leaving behind nuclei of white blood cells from which DNA can be extracted. However, this technique will result in the loss of cytoplasmic DNA to the supernatant, and hence will not be able to extract mitochondrial DNA (B11). 

COLI PRODUCES SEVERAL COPIES OF ITS OWN DNA AS WELL AS DNA INSERTED INTO PLASMID. 

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