LABORATORY IX

CONTENTS:

1.     Gene cloning

a)        Polymerase Chain Reaction and analysis by agarose gel electrophoresis

b)        Plasmid DNA isolation and DNA quantitation

c)        Restriction Enzyme digestion of plasmid DNA

d)        Vector and Insert Ligation

e)        Preparation of competent cells

f)         Transformation of E.coli with standard plasmids, Calculation of transformation efficiency

g)        Confirmation of the insert by Colony PCR and Restriction mapping

2.      Expression of recombinant protein, concept of soluble proteins and          inclusion body formation in E.coli, SDS-PAGE analysis

3.      Purification of His-Tagged protein on Ni-NTA columns

4.      Random Primer labeling

5.      Southern hybridization

6.     Genetic Transfer-Conjugation, gene mapping