Techniques employed in recombination DNA technology: Isolation and purification of DNA, Gel electrophoresis (Gel, Agarose), DNA sequencing, Southern and Northern blotting, PCR and its application
Isolation and purification of DNA:- 1. The extraction buffer:- This includes - i. Detergent:- Such as cetyl trimethyl ammonium bromide(CTAB) or SDS which disrupts the membranes. ii. Reducing agent:- Such as Beta mercaptoethanol which helps in denaturing proteins by breaking the disulfide bonds between the cysteine residues and for removing the tanins and polyphenols present in the crude extract. iii. Chelating agent:- Such as EDTA which chelates the magnesium ions required for DNase activity. iv. Buffer:- which is almost always Tris at pH 8. v. Salt:- Such as sodium chloride which aids in precipitation by neutralizing the negative charges on the DNA so that the molecules can come together. 2. Phenol chloroform extraction:- > Nucleic acid solutions commonly contain undesirable contaminants that are chiefly made of proteins. A classic method of purifying is phenol –chloroform extraction by which the the nucleic acid solution is extracted by successively washing with...