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Procedure

  1. Bacterial Transformation
    • IMPORTANT: In addition to wearing the appropriate personal protective equipment, be sure to take care to keep your face away from suspension cultures, and to avoid inhaling reagents. Do not touch your face while performing the experiment. Always wash your hands before and after every experiment.
    • When you are ready to safely begin, check to make sure you have a tube or tubes of competent E. coli cells in your ice bucket.
    • Then, transfer 50 µL from one of these tubes to each of two new micro-centrifuge tubes, one labeled '–', and one labeled '+'.
    • Next, pipet 2 µL of p-GREEN plasmid into the plus tube and flick the tube gently to incorporate the plasmid throughout the bacterial solution. HYPOTHESES: The experimental hypothesis is that on plates containing no antibiotic, bacteria with or without the plasmid can survive, and so both will grow lawns of bacteria. Conversely, on the plates containing the ampicillin antibiotic, only the bacteria successfully transformed with the resistance plasmid will grow, whereas the minus plasmid plates will contain no bacteria. The null hypothesis of this experiment is that there will be no difference in bacterial growth between the plates.
    • Incubate both tubes on ice.
    • After 30 minutes submerge both tubes 3/4 of the way into the water of a 42 °C water bath for 30 seconds.
    • At the end of the water bath incubation, place the tubes back on ice for 5minutes, then add 950 μL of room temperature SOC media to each tube.
    • Incubate the tubes in a shaker at 37 °C for 60 minutes at 250 rpm.
    • While the tubes are incubating, label the bottoms of one LB and one LB/ampicillin, or AMP plate, “+ plasmid”, and the bottoms of one LB plate and one LB/AMP plate “- plasmid”.
    • At the end of the incubation, pipet 50 μL from the “+ plasmid” cell tube onto both the LB/AMP plus plate and the LB plus plate.
    • Then, with a fresh tip, add 50 μL of the bacteria that were not exposed to the plasmid to each of the minus plates.
    • Then, spread each plate using a fresh pipette tip.
    • Cover the plates with their lids, and then seal them with laboratory film.
    • Then store the plates upside down in a 37 °C incubator for 24 hours.
  2. Results
    • The day after the transformation, observe which plates have bacteria, and how the bacteria are distributed over each plate.
    • Record the number of colonies and the color of the colonies for different conditions. Click Here to download Table 1
    • To determine the initial mass, IM, of the plasmid, first multiply the plasmid concentration from the stock tube of p-GREEN by the volume transferred into the experimental tube.
    • To calculate how much DNA is spread onto each plate, divide the volume of bacterial suspension spread onto each plate by the volume of the suspension in the tube, and multiply this fraction by the initial mass of plasmid used.
    • To determine the transformation efficiency, count all of the visibly distinct colonies on the plus plasmid LB/AMP plate, and divide this number by the mass of plasmid spread. NOTE: The unit for transformation efficiencies is cfu/μg of plasmid DNA.
    • Record all of the results for each plate in the table and analyze them.
    • Observe any differences between the colonies and make hypotheses to explain this observation. Consider the factors that may affect the transformation efficiency of competent bacteria.
    • NOTE: You will have also noticed that each plate had a different outcome. The minus plasmid and plus plasmid LB plates should have grown a lot of bacteria across their plates. The minus plasmid LB/AMP plate should have no bacterial growth. And the plus plasmid LB/AMP plate should have grown bacterial colonies that fluoresced green.

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