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 ResistanceBeta-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”

Follow page 2-17 !!!!!

Control25 ul buffer

300 ul cells

pGAL DNA25 ul plasmid

300 ul cells

Flow Chart for Transformation

Use transferpipette to placeENTIRE cell suspensionin each tube

X-Gal Amp/XgalAmp/X-Gal

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

Control DNA

Agar plate with drops of transformed cellsPlace 10 drops of 25 ul each on the plate

Cell spreaderGently spread across surface

Let plate sit 10-15 min.CoverIncubate at 37oC overnight

Plating of transformed bacteria

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

Makes bacteriaresistant toampicillin

Protein Product:Beta-galactosidase

Cleaves sugars,Utilizes X-gal toproduce a bluecolor

Plasmid= a Circular, Independently-replicating Piece of DNA

pUC8

Lac Z gene

2665 bp

Amp r

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. White2. Blue3. Either white or blue

4. Both white and blue5. 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. White2. Blue3. Either white or blue

4. Both white and blue5. Neither white nor blue

Next lab: Transformation Efficiency is Determined

Our experiment uses:

DNA concentration: 0.025 ugRecovery Volume: 1 mlPlating Volume: 0.25 ml

Number of Transformantsamount of DNA used (ug)

Final Recovery Volumevolume plated (ml)

X =

Number of Transformants per ug