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

General Laboratory Practices Wear disposable gloves, especially when handling cells, DNA, and agarose gels Obtain reagents from front bench or cart Keep sterilized materials clean, minimize contact with air and hands Dispose of waste material in red biohazard bags unless otherwise instructed Read laboratory instructions before doing the experiment

TRANSFORMATION Uptake of DNA from the external environment In this experiment, we will add plasmid DNA to bacterial cells. Those that take up the plasmid will be transformed. The incoming DNA alters their genotype and observable phenotype.

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

How do we know if transformation occurred? You must “plate” your transformed bacteria. We identify transformed cells by selectable markers. Ampicillin Resistance Beta-galactosidase activity (causes color change)

Group materials Each group (4 persons) Micropipettors + Tips Plasmid DNA Buffer Competent Cells Recovery broth 3 agar plates: X-gal only (1), Amp + X-gal (2) 3 transfer pipets 1 “yellow plater”

Flow Chart for Transformation Follow page 2-17 !!!!! Flow Chart for Transformation 300 ul cells 300 ul cells Use transfer pipette to place ENTIRE cell suspension in each tube Control 25 ul buffer pGAL DNA 25 ul plasmid

Flow Chart for Transformation Follow page 2-17 !!!!! Flow Chart for Transformation Incubate 10 min. on ice Incubate 42 oC for 90 seconds Place on ice for 1 minute Add 0.7 ml recovery broth Incubate at 37 oC for 30 min Add 0.25 ml of cells to each plate DNA Control Amp/X-Gal X-Gal Amp/Xgal

Plating of transformed bacteria Cell spreader Gently spread across surface Let plate sit 10-15 min. Cover Incubate at 37oC overnight Agar plate with drops of transformed cells Place 10 drops of 25 ul each on the plate

Micropipetting Select the appropriate sized pipettor and dial in the volume needed Use the end of the pipettor to pick up a disposable tip, use your hand to tighten the tip only at the largest end Push the plunger to the first stop, insert the tip beneath the liquid and slowly release the plunger Insert tip into receptacle (either against tube wall or beneath surface of liquid), push plunger to second stop to release contents Release used tip into biohazard bag

Selectable Markers on the Plasmid Protein Product: Beta-lactamase pUC8 Amp r Makes bacteria resistant to ampicillin Protein Product: Beta-galactosidase 2665 bp Lac Z gene Cleaves sugars, Utilizes X-gal to produce a blue color Plasmid= a Circular, Independently- replicating Piece of DNA

Applying Your Knowledge Cells that take up the pUC plasmid should grow on plates containing 1. Ampicillin 2. X-gal 3. Both Ampicillin + X-gal 4. Neither Ampicillin nor X-gal

Applying Your Knowledge What color will colonies of Control cells (without the pUC plasmid) be on a plate containing X-gal? 1. White 2. Blue 3. Either white or blue 4. Both white and blue 5. Neither white nor blue

Applying Your Knowledge What color will colonies of Transformed cells (with the pUC plasmid) be on a plate containing X-gal? 1. White 2. Blue 3. Either white or blue 4. Both white and blue 5. Neither white nor blue

Next lab: Transformation Efficiency is Determined Number of Transformants amount of DNA used (ug) Final Recovery Volume volume plated (ml) X = Number of Transformants per ug Our experiment uses: DNA concentration: 0.025 ug Recovery Volume: 1 ml Plating Volume: 0.25 ml