Bio110AntibioticResistanceLabManual2013_

Experimentpartccountingbacterialculturesgrowingonkanam

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Unformatted text preview: king in a group of 6 at your bench. 2. Each bench will have a total of six tubes containing the PCR reaction mix. The tubes will be color‐ coded. Table 1 below shows the ingredients of each tube. Three tubes will have primers only (the orange, blue and yellow tubes). These are the tubes to which you will add some of your colonies. The three other tubes will have primers with a control plasmid (the red, green and pink tubes). You will NOT add anything to these tubes. 3. Identify and number three antibiotic‐resistant colonies growing on plates labeled “K.” (These can be from the same plate or from different plates generated from the bacterial sample with the same code letter (i.e., from the same chicken farm)). 4. Touch a white pipette tip into colony #1 and then dip it into the orange tube. (Try and get a bit of the colony to release, but don’t worry if this doesn’t happen. There will be sufficient bacterial DNA for the PCR reaction to be effective). Dispose of this contaminated pipette tip into the red biohazard bag on your bench, and be sure to snap closed the orange tube’s lid. 5. Take a new pipette tip and repeat the same process into the blue tube with colony #2. Dispose of this contaminated pipette tip into the red biohazard bag on your bench, and be sure to snap closed the blue tube’s lid. 6. Repeat the process again for the yellow tube with colony #3. Dispose this contaminated pipette tip into the red biohazard bag on your bench, and be sure to snap closed the yellow tube’s lid. 7. The red, green, and pink tubes will serve as positive controls; do NOT add anything to these tubes. 8. Load your six tubes into the PCR machine. Your TA will help you run the PCR reaction. 7 Table 1. The following table summarizes what is in each of the tubes; the darkly outlined cells represent the tubes you will add your colony samples to as described above. Tube Color PCR Primer Control Plasmids Colonies from your chicken farm’s bacterial plate “K” Your farm’s name: A B C A B C Orange + -- -- -- -- -- + Blue...
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This note was uploaded on 02/06/2014 for the course BIO 110 taught by Professor Hass during the Fall '11 term at Penn State.

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