Bacterial Transformation Lab
In this experiment we transformed a strain of E. Coli bacteria without antibiotic resistance with plasmid DNA. This plasmid produces a fluorescent green glow under black light due to the gfp(green fluorescent protein) as well as antibiotic resistance. E. Coli cells will be plated on an agar medium, some with and some without the antibiotic ampicillin. Only bacterial cells that contain the plasmid will survive the ampicillin and produce the green glow.
This experiment allowed us to observe the process of bacterial transformation. I believe that only a small percentage of the cells will transform and the gfp plasmid will be most apparent in the agar plate containing both the plasmid and ampicillin.
Materials and Procedures:
4 agar plates
E. Coli bacteria
Luria Recovery Broth
Hot water bath
2 microcentrifuge tubes
label one microcentrifuge “+DNA” this will contain the gfp plasmid DNA label the second tube “-DNA” this will be the control tube without gfp plasmid DNA label one agar plate LB-, the second LB+, the third LB/amp-, and the last one LB/amp+ (amp plates will contain ampicillin and + plates will contain gfp plasmid) using a 1-ml pipette, transfer .25ml of ice cold CaCl2 solution into each tube Transfer E. Coli colonies into each of the tubes using a sterile toothpick Add .10ml of the gfp plasmid to the tube labeled “DNA+”
Incubate the tubes on ice for 15 minutes
place both tubes into a 42 degree hot water bath for 90 seconds allowing the heat shock to let the plasmid DNA enter the cells Immediately return both tubes to the ice bucket and incubate for2 minutes Using a sterile pipette, add .25ml of luria broth to each tube and mix Incubate cells in a 37 degree water bath for 30 minutes to recover Using a sterile pipette, transfer .25ml of the “DNA-” mixture to the plate labeled LB-...
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