Transcript PPT File
DNA Transformation Lab
E Coli with PCU
Avery–MacLeod–McCarty
experiment
• Showed DNA is the substance that causes
bacterial transformation
Hershey- Chase
• Showed DNA is the genetic material
Propagating DNA in a host cell
Requires a vector
1) Plasmid
2) Phage virus
Plasmids
Circular molecules of double stranded
DNA that self replicating.
They code for cell functions but are not
essential
Exchange of genes
Prokaryotes exchange genes for diversity
(asexual)
1) Transformation
DNA is released into surrounding medium
Recipient cells incorporate it into
themselves from the medium
2) Transduction
A phage virus attaches to bacterial cell
and transfers its DNA into the bacterium
Similar???
3) Conjugation
Plasmids travel between touching cells
R factors
Plasmids that contain resistance to
antibiotics
Transformation in streptococcus
pneumoniae in mice
Competent cells
Cells that can be transformed by DNA
Some can become competent by certain
environmental conditions
EX. E-coli
E. Coli = Escherichia coli
Normally harmless bacteria in your gut
E. Coli can be artificially transformed
by exposure to calcium chloride solution
and thermally “shocked”
To become receptive to foreign plasmid
In this lab we will perform
transformation of E Coli
pUC8 = Ampicillin Resistant plasmid will
be inserted into E coli
LAB
Sterile technique!!
Don’t touch anything
Lift Petri lid partially, pour/swab etc.
Immediately reclose
Open instruments and use immediately
Do NOT touch working end
Close quickly
Incubation
Incubate plates UPSIDE DOWN
Antibiotic
Ampicillin – Kills bacteria
After mixing keep in freezer
Thaw 30 mins. prior
Needs
Thermally shock to make E Coli competent
to receive plasmid
ICE BATH followed by
Warm Water bath
Bacteria Colonies
Lawn – all together not separate
4 plates
Luria Broth Luria Broth +
AMP Broth AMP Broth +
No antibiotic, no plasmid
No antibiotic, With plasmid
antibiotic, No plasmid
antibiotic, With plasmid
What do you expect
Luria Broth -
No antibiotic, no plasmid
Normal growth = lawn of bacteria
Luria Broth +
No antibiotic, With plasmid
Normal growth = lawn of bacteria
AMP Broth -
antibiotic, No plasmid
NO growth, all killed
AMP Broth +
antibiotic, With plasmid
Individual Colony growth of transformed resistant
bacteria
results
Calculating efficiency of
transformation
Total mass of plasmid used
Total mass = volume X concentration
Volume = 10 µl
Concentration = 1 µg/100µl
= 0.1 µg plasmid
Total volume of suspension
Volume calcium
.25 ml =
chloride (chilled)
250µl calcium chloride
Volume Plasmid
10 µl Plasmid
Volume Luria broth .25 ml =
250 µl luria broth
TOTAL Volume of Total = 510 µl
suspension
Fraction of suspension put on plate
µl on plate/total volume
.1 ml put on plate
=100 µl
Total volume = 510 µl (from above)
100 µl x 510 µl =
0.196 µl put on plate
Total mass of plasmid in fraction
(mass of plasmid x fraction on plate)
Mass of plasmid = 0.1 µg
Fraction on plate = 0.196
.1µg x 0.196 =
Total mass of plasmid on plate = 0.0196 µg
Number of colonies
per µg of plasmid
(# colonies counted/mass of plasmid put
on plate)
Count = #
Mass of plasmid = 0.0196 µg
Colonies per µg plasmid = …………