Bio5LAManual12f

After growth each lab group will score the number of

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Unformatted text preview: cells do not spontaneously take up DNA. Therefore prior to this laboratory, E. coli cells were treated with CaCl2 to enhance DNA uptake; these cells are “competent” for DNA uptake. Using the procedure described below, each group of students will mix DNA with the competent E. coli cells. Any cell that takes up the plasmid DNA and maintains it to express ampicillin-resistance is called a transformant. One third of the cells will be plated on ampicillin plates to determine the number of transformants. One third of the cells will be plated on Luria broth agar (LB) plates as controls. The third portion of cells will be serially diluted and plated on antibiotic-free media. The latter will be used to determine the total number of bacterial cells that were available for transformation. After growth, each lab group will score the number of transformants obtained and determine the total number cells available for transformation. These figures will be used to calculate transformation efficiency which, for our purposes, is the number of transformants obtained divided by the number of cells available for transformation. *Note: There is a flow chart included on page 5 of this exercise to aid you in performing this experiment. Be sure to fill in each value, and time component prior to your laboratory period. Biology 05LA – Fall Quarter 2012 Lab 7 – Page 2 To demonstrate bacterial transformation, each group of students will receive: 1. 2. 3. 4. 5. 6. 7. 10 drops bacterial cells (Escherichia coli) pUC118 plasmid DNA two ampicillin-containing petri plates (AMP, blue stripe label) five petri plates containing media without antibiotics (LB, unlabeled) sterile saline for bacterial cell dilutions three sterile 15-ml dilution tubes sterile L broth (bacterial growth medium) Hazard: You will be using a plasmid DNA that carries an antibiotic resistance gene. While this poses no danger to you or your lab partners, it is important that we do not introduce the plasmid into nature. For this reason, we will be handling this DNA and the bacteria in accordance with Federal Guidelines for Recombinant DNA. It is critical that you dispose of all items that come in contact with DNA or bacteria in the specially designated containers. All items will be autoclaved prior to disposal. Procedure: Be advised that accurate results in this experiment can only be obtained if the following two cautions are heeded: First, all measurements must be made as precisely as possible. Second, when transferring the prescribed volumes of the bacterial cell suspensions, care must be taken to make sure that the source container of cells is completely suspended before the transfer is made – your TA will demonstrate the correct procedure for doing so. 1) Allow a microfuge tube containing 10 drops competent E. coli cells to thaw on ice. 2) Using a sterile long-tipped Pasteur pipette fitted firmly into a blue pipette pump (be careful here not to break the pipette by pushing too hard) slowly withdraw most of the volume of the thawed cells into the pipette. As you pull the pipette from the microfuge tube, briefly touch the tip of the pipette against the inner wall of the tube to clear any cell suspension from the outside of the pipette tip. Then, carefully transfer 3 drops (approx. 0.1 ml) of the cell suspension into each of two sterile microfuge tubes and close their caps. Return any cells still in the pipette to the tube of unused cells, cap the tube, and set it aside for later use. 3) Label one of the two tubes that you just filled "+", and the second tube "-". The "+" stands for plus plasmid DNA added; the "-" stands for no plasmid DNA added. 4) Your TA will add 1 drop pUC118 plasmid DNA to the "+" tube. (This is being done to ensure the accuracy of the DNA measurement.) Gently re-suspend the cells. 5) Add 1 drop sterile saline to the "-" tube. Gently re-suspend the cells. 6) Incubate both the "+" and "-" tubes on ice for at least 20 minutes. 7) During this incubation you should, get ready to determine the number of viable E. coli cells available for transformation. To do this, you will make serial dilutions starting with 3 drops of the remaining E. coli cells: a) Each group of students should collect three sterile culture tubes, and label them "A", "B" and "C". b) To tube "A", add 9.9 ml of sterile saline. To tubes "B" and "C", add 9.0 ml sterile saline. c) Set these tubes aside and continue the transformation experiment started earlier. Biology 05LA – Fall Quarter 2012 Lab 7 – Page 3 8) Resuspend the cells in your "+" and "-" tubes and place the tubes into the 42oC water bath for 2 minutes*. Remove the tubes from the water bath promptly after this incubation. * Be careful not to exceed this time!! Long periods at high temperature will kill E. coli. The o incubation at 42 C is called a “heat shock: and makes the plasma membrane more fluid so that DNA can be taken up by the ce...
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This note was uploaded on 08/27/2013 for the course BIO BIOL05LA taught by Professor Abbottl during the Fall '12 term at UC Riverside.

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