Lab6_Wilkerson - Christopher Wilkerson 1 21:54:25...

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Unformatted text preview: Christopher Wilkerson 1 21:54:25 Transformation & Miniprep Objective : The purpose of this lab is to use the chemically competent cells that we created to learn how to transform bacteria using various plasmids. The transformed cells will then be harvested and grown for DNA extraction and purification using miniprep. One plasmid will be plated using different concentrations to test the efficiency of the competent cells Also we will test the cells for contamination of mold or naturally ampicillin resistant bacteria. Lab Protocol : Plasmid Transformation and Miniprep - The three competent cell 50 L aliquots were first taken out of an -80 C freezer and thawed on ice. The cell tubes were then labeled by the name of the plasmid that would be used in the transformation reaction. The plasmids were as follows: mCherry, tetR, and GFP. Three aliquots of sterile SOC media were taken out of a freezer and placed into a 37 C incubator. Five LB-amp plates were also taken out of the freezer and placed into the incubator. Once the competent cells thawed 50 ng of each plasmid was micropipetted into its own tube of cells. The concentration of the three plasmids and the subsequent volumes used were as follows: tetR 85.2ng/ L , 0.587 L mCherry 147.06ng/ L , 0.340 L GFP 85.2ng/ L , 0.587 L The tubes were then incubated on ice for 30 minutes. After incubation the cells were heat shocked in a 42 C water bath and immediately placed back on ice for two minutes. 950 L of room temperature SOC media were transferred into each tube. The tubes were then shaken horizontally in a 37 C incubator for one hour. After the incubation the cells were then spread plated on the LB-amp plates that had been incubating. This process began with putting five sterile colirollers onto each plate. 200 L of both the mCherry transformed bacteria and Christopher Wilkerson 2 21:54:25 the tetR transformed bacteria were then micropipetted onto their own plates and were spread on the plate by swirling the colirollers around the plates until the bacteria had been spread evenly. For the GFP transformed bacteria, three different concentrations of the bacteria were prepared and then plated using the same technique. The first two had 10 L and 100 L of original plasmid bacteria and 190 L and 100 L of LB media to keep the final volume 200 L. The third began with 800 L that was centrifuged at 3000 rpm for one minute. The L that was centrifuged at 3000 rpm for one minute....
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This note was uploaded on 06/23/2008 for the course CH 204 taught by Professor Leytner during the Spring '08 term at University of Texas at Austin.

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Lab6_Wilkerson - Christopher Wilkerson 1 21:54:25...

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