Learning Module - Experiments 3A & 3B

Preparation of bacteriophage and plasmid DNA.

Mother Nature was used to make DNA-containing particles in Experiments 2A and 2B.

In Expt. 2A we grew XL-1 Blue cells containing the pTrc99A expression vector

In Expt. 2B we propagated lamda ZAPII/topAcycB

The expression vector pTrc99A contains a gene for Ampicillin resistance

Chloramphenicol inhibits teh peptydyl transferase activity of bacterial 50S ribosomal subunit

pTrc99A DNA is prepared in Experiment 3A

Specific additions are made in plasmid mini-preps

Bacteriophage DNA is isolated in Experiment 3B

Tris/EDTA buffers the solution and removes divalent cations which are needed by nucleases

Potasium acetate precipitates the SDS-protein complex

Ethanol causes DNA to precipitate

These DNAs will be used to subclone the topA/cysB insert in Experiment 4B

Subcloning the tipAcysB genes