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 = …………