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