Transformation Flashcards
What is the purpose of transformation?
To change an organism’s traits/phenotype
What is the purpose of CaCl2?
To move the plasma DNA through the cell membrane by neutralizing the negative charges on the cell membrane
What is the purpose of heat shock?
Opens phospholipid bilayer to allow plasmid to go inside the cell
At what temperature are the bacteria heat shocked?
42 degrees celsius
The purpose of incubating bacteria on ice?
Helps stabilize cell membrane and promotes stable binding of DNA to bacterial surface by closing phospholipid bilayer
Why is arabinose used?
Activates AraC which activates GFP gene which causes the fluorescence in the agar medium
Transformation efficiency calculations.
of colonies/ conc. of plasmid DNA x 1/ plate volume
The procedures of transformation.
- Label tubes P+ and P- and cells - and pGLO+
- Add bacteria using loop to tubes
- Pipet solution to mix
- Place in ice bath for 20mins
- Pipet 200 microliters from - and place in +
- Incubate tubes in 42 degrees celsius water bath for 90 secs to heat shock
- Place tubes back on ice
- Recover by adding 200 microliters of LB to each tube
- Incubate at 37 degress celsius for 30 mins
- Transfer bacteria to designated agar plate
What is recovery broth and what is its purpose?
A nutrient rich medium used to support the recovery of transformed cells from its strenuous process
What plasmid was used in the experiment.
pGLO plasmid
Explain the results of the 4 plates plated.
LB- : growth (control)
L Amp-: no growth (due to absence of ampicillin)
L Amp+: growth but no glow (growth due to antibiotic resistance but no glow due to lack of arabinose to active glow gene)
L Amp Arabinose: growth and glow (arabinose activated glow gene)
