Laboratory: Bacterial Transformation Introduction of plasmid (pGal) DNA into E. coli

This laboratory is The first part in a series of 3 experiments: Plasmid Transformation Plasmid Isolation Plasmid Mapping

Principle of transformation experiment Genotype determines phenotype

Your plasmid contains A ampr gene And a lac z gene Amp r pGal

Lac Z gene Codes for beta-galactosidase Beta-galactosidase is secreted by the transformed E. coli Beta-galactosidase utilizes the substrate “X-gal” to produce a blue color

Amp resistance gene Beta-lactamase secreted extracellularly Beta-lactamase inactivates ampicillin

How to transform cells Competent bacterial cells are required Introduction of plasmid DNA + bacteria “Heat Shock” to increase uptake of DNA

How do we know if transformation occurred? You must “plate” your transformed and allow your bacteria to grow Identify transformed DNA by a “selectable marker”.

Group materials Each group Plasmid (pGal) DNA Buffer Recovery broth 3 agar plates 3 transfer pipets or use micropipettors 2 “yellow platers”

Laboratory Protocol Always wear gloves Reagents are on cart or front bench Ice is on front bench Divide into groups Dispose of material in red container in front of room

Plating of transformed bacteria Cell spreader Gently spread across surface Let plate sit 10-15 min. Cover Incubate 37 overnight Agar plate with drops of transformed cells

Cart and lab bench has the supplies: come get your supplies 9-10 groups! Each lab group should have at their bench 1 plate labeled X-GAL 2 plates labeled “AMP/X-GAL” 1 microtest tube labeled “pGal DNA” 1 microtest tube labeled “Control Buffer” 1 microtest tube labeled “Cells for DNA” 1 microtest tube labeled “Cells for Control” 1 microtest tube labeled “Recovery” 4 sterile 1 mol pipets 1 inoculating loop

Next lab: Transformation Efficiency is Determined # of transformants/ug of DNA x volume at recovery (ml)/volume plated (ml)= # of transformants per ug of DNA Our experiment uses: DNA concentration: 0.025 ug Recovery Volume: .68 ml Plating Volume: 0.25 ml