Bio110AntibioticResistanceLabManual2013_

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Unformatted text preview: hem to the storage slots inside the gel box. 6. Once you have relocated the dams, pour electrode buffer (1xTAE) into the gel electrophoresis unit. Be sure the anode and cathode sides are full and the gel is completely covered by the buffer. 7. Gently remove the comb by pulling straight up. Create a heading in your notebook: “Experiment Part D: Identifying Gene for Kanamycin Resistance” Loading and Running the Gel: Record any intentional or unintentional changes in procedure in your science notebooks 1. Load 5 μl of the PCR DNA ladder as a molecular size standard into the first well. You will find this in a tube, labeled ladder, found in an ice bucket in the lab room. 2. Add 2 μl of 6x loading dye into each of the six tubes containing your samples. Mix thoroughly by gently pipetting up and down in the tube after adding the loading dye. This dye contains glycerol, a dense substance that will allow your sample to sink to the bottom of the well, and bromophenol blue and xylene cyanol ff, which will allow you to see how far your sample has electrophoresed. 3. You will load 15 μl from your six colored tubes into the next six wells, in the order indicated in Table 2. 4. Once all your samples are loaded, place the lid firmly on the top of the unit. Insert the electrodes into the power supply and turn on the power to 160 volts (or as otherwise directed by your TA). 5. Once the bromophenol blue tracking dye (purplish‐blue) has migrated at least ½ of the length of your gel, put on disposable gloves, turn off the power, and carefully remove the gel. 6. Place the gel into the tray at your bench and carry it to the UV light box so you can view and photograph your gel. 10 Table 2 provides a summary of what was loaded into each of the wells of the agarose gel: Lane/Well Sample Contents 1 DNA ladder Molecular weight standard 2 PCR sample 1 (Orange tube) Plasmid DNA from Colony 1 3 PCR sample 2 (Blue tube) Plasmid DNA from Colony 2 4 PCR sample 3 (Yellow tube) Plasmid DNA from Colony 3 5 PCR sample 4 (Red tube) Standard of Plasmid A 6 PCR sample 5 (Green tube) Standard of Plasmid B 7 PCR sample 6 (Pink tube) Standard of Plasmid C Photographing Gels: Record any intentional or unintentional changes in procedure in your science notebooks 1. Place gel on UV light box in the middle of the blackened glass (this is the UV light sourc...
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