Transcript Transformed

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)
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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 !!!!!
Flow Chart for Transformation
300 ul cells
Control
25 ul buffer
300 ul cells
Use transfer
pipette to place
ENTIRE cell suspension
in each tube
pGAL DNA
25 ul plasmid
Follow page 2-17 !!!!!
Flow Chart for Transformation
•Incubate 10 min. on ice
Control
•Incubate 42
oC
DNA
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
X-Gal
Amp/Xgal
Amp/X-Gal
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
Makes bacteria
resistant to
ampicillin
Amp r
pUC8
2665 bp
Protein Product:
Beta-galactosidase
Lac Z gene Cleaves sugars,
Utilizes X-gal to
produce a blue
color
Plasmid= a Circular, Independentlyreplicating 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 X
amount of DNA used (ug)
Final Recovery Volume
=
volume plated (ml)
Number of Transformants per ug
Our experiment uses:
DNA concentration: 0.025 ug
Recovery Volume: 1 ml
Plating Volume:
0.25 ml