Insert Purification, Plasmid Ligation and Transformation

 

PLASMIDS FOR SUBCLONING

 

Notes


It is not generally necessary to dephosphorylate cut plasmids for subcloning. However for library production it is essential to minimise plasmid religation and this method would not be applicable.
 
 

SUBCLONING RESTRICTION FRAGMENTS

 

Notes


Obviously gel purified inserts will result in plasmids bearing the required product. However sometimes it is desirable to ligate a mixture of restriction products, such as those from a recombinant l phage DNA digest, resulting in plasmids bearing a variety of restriction products. It is simple to digest such target materail with enzyme(s) of choice and then extract and precipitate the reaction mixture (as with the production of cut plasmids) without any gel purification step, and then use directly in ligation reaction as described above.

Plasmids and digests may also be double digested or sequentially digested and filled-in after digestion to generate partially filled or blunt ends.
 

Reagents


LB media; 10g tryptone, 5g yeast extract, 5g NaCl

LB agar; LB media with 10g/l agar

50X TAE buffer; 2M TRIS/Acetic acid, pH 8.0, 250mM sodium acetate, 50mM EDTA