Bacterial Transformation Lab Report
The plasmid pGLO contains an antibiotic-resistance gene, ampR, and the GFP gene is regulated by the control region of the ara operon. Ampicillin is an antibiotic that kills E. coli, so if E. coli, so if E. coli cells contain the ampicillin-resistance gene, the cells can survive exposure to ampicillin since the ampicillin-resistance gene encodes an enzyme that inactivates the antibiotic. Thus, transformed E. coli cells containing ampicillin-resistance plasmids can easily be selected simply growing the bacteria in the presence of ampicillin-only the transformed cells survive. The ara control region regulates GFP expression by the addition of arabinose, so the GFP gene can be turned on and off by including or omitting arabinose from the culture medium.
The purpose of this lab was to understand bacterial transformation, how it occurs, and to make DNA glow.
If the transformed E. coli is mixed with the ampicillin resistance gene, it will be able to grow in the ampicillin plates, but the non-transformed E.coli will not.
Two microcentrifuge tubes
500 uL of ice cold 0.05 CaCl2
E. coli bacteria
A sterile plastic loop
A sterile P-20 micropipette
10 uL of pAMP solution
A water bath
500 uL of Luria broth
A spreading rod
Day before lab
1. Streak E. coli host cells for isolation.
2. Prepare six source plates.
Day of lab
1. Get two microcentrifuge tubes, which should each contain 200 uL of cold CaCl2 solution. Label one tube with your initials and a (+) and the other tube with your initials and a (-). 2. Transfer 2-4 large colonies using a sterile plastic loop to each microcentrifuge tube and completely resuspend. Do not transfer any agar. Put the tip of the loop into the CaCl2 solution and spin until there is not any cells on the loop. 3. Close each of the tubes and put them in ice.
4. Ask your teacher to...
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