Pblu Lab Report

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Purpose In this lab we are trying to get a broader understanding of the transformation of bacteria by exposing them to pBLU plasmids. Introduction Transformation is the manipulation of a bacterial cell's DNA in order to alter the cell's genotype or phenotype by absorbing free DNA from its surroundings. This can result in a nonpathogenic bacteria becoming pathogenic by absorbing the DNA of a broken open or dead pathogenic bacteria. In our case it is taking in the pBLU plasmid. A plasmid is a spherical self-replicating DNA molecule that is not actually a part of the bacterial cell but can integrate itself into the bacterial chromosome. While it is not required for the living and reproduction of the bacterial cell plasmids can provide advantages in stressful environments such as the ability to break down X-Gal in this experiment. Procedure 1. Mark one sterile 15-mL tuba "+pBLU;" mark another "-pBLU." (Plasmid DNA will be added to +BLU tube; no will be added to –BLU tube.) 2. Use a sterile transfer pipet to add 250 μL of ice-cold calcium chloride each to +BLU tube and –BLU tube. 3. Place tubes on ice. 4. Use a sterile plastic inoculating loop to transfer one or two large colonies of E. coli cells from the starter plate to the +BLU tube. a) Be careful not to transfer any agar from the plate along with cell mass. b) Immerse loop tip in calcium chloride solution, and vigorously tap against wall of tube to dislodge cell mass. Hold tube up to light to observe cell mass fall off loop. 5. Immediately suspend cells in +BLU tube by repeatedly pipetting in and out, using the sterile transfer pipet. Holed tube up to light, and carefully inspect to see that suspension is homogenous. No visible clumps of cells should remain in the tube or be "lost" in the bulb of the transfer pipet. 6. Return +BLU tube to ice. Transfer a second mass of cells to –BLU and suspend as

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