Bacterial Transformation Lab .pdf - Bacterial...

Bacterial Transformation Lab Questions
Introduction Paragraph: In this lab, the effects of genetic modification will be observed using
E. Coli bacteria. A control group of unchanged DNA and DNA with ampicillin will also be
observed in the experiment. In the end we will be able to determine the changes genetic
modification played in the bacteria’s reproduction and phenotype. Observations from this lab can
help analyze the results that other experiments involving GMOs and genetic engineering
produce.
Problem Statement: What is the effect of genetic modification on E. Coli using genes from
jellyfish and ampicillin related substances?
Hypothesis : If the bacteria receives a ampicillin resistant trait then it will be able to reproduce in
a high manner, because the ampicillin will not be able to kill it.
Independent Variable : The plasmid in the DNA
Dependent Variable : The amount of bacteria colonies that have grown.
Control : The plate of LB that only used original bacteria and no genetic modification.
Materials :
- E. Coli bacteria
- 2 LB agar plates
- 2 LB/amp (ampicillin) agar plates
- CaCl2
- LB nutrient broth
- Sterile inoculation loops
- Pipettes
- DNA plasmid
- Microcentrifuge tubes for the DNAs and solutions
- Ice bath beaker
- Incubator
- Aprons
- Goggles
- Ultraviolet light source
- Timer
- 10% household bleach
Procedure :
1. Add 500 μl of CaCl2 into the Negative DNA centrifuge.

2. Using a toothpick, collect 15 colonies of E. coli bacteria and swirl it around in the
centrifuge made above.
3. Then using a clean pipette, transfer 250 μl of the negative DNA to the positive DNA
centrifuge.
4. Using the pipette again, transfer the positive DNA into the centrifuge containing 10 μl
pGFP.
5. Incubate these centrifuges in an ice bath for approximately 10 minutes.
6. Remove tubes from ice bath and incubate at a temperature of 42°C for approximately 90
seconds.
7. After this, add 250 μl of “recovery broth” to both sets of centrifuges.