Download Transformation laboratory

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
This is in your lab manual!
SUMMARY
•Incubate 10 min. on ice
Control
•Incubate 42 C for 90 seconds
Treatment
•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
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