Laboratory: Bacterial Transformation Introduction of plasmid DNA into E. coli
This laboratory is The first part in a series of 3 experiments: Plasmid Transformation Plasmid Isolation Plasmid Mapping
Transformation A process of plasmid DNA uptake In our experiment the plasmid is: extrachromosomal
Transformation experiment illustrates: Genotype determines phenotype
Plasmid DNA How will the phenotype of the E. coli be changed?
Plasmids have selectable markers to detect change: Color alteration of colonies Antibiotic resistance
Let’s look more closely at “our” plasmid Amp r pGal Lac Z gene
What are characteristics of the lac Z gene?
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
What are characteristics of ampr gene?
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
Bacterial Tranformation Protocol
Experimental overview: Please refer to your lab manual.
Group materials Each group Plasmid DNA Buffer Recovery broth 3 agar plates 3 transfer pipets or use micropipettors 2 “yellow platers”
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
SUMMARY This is in your lab manual! Treatment Control Amp/X-Gal X-Gal Incubate 10 min. on ice Incubate 42 C for 90 seconds Place on ice for 1 minute Add 0.75 ml recovery broth to control and treatment tubes Incubate at 37 C 15-30 min streak 10 drops of cells evenly Treatment Control Amp/X-Gal X-Gal Amp/Xgal
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