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To confirm that the ''E coli'' bacterial colony you picked has ''lys-2'', we are going to do a '''colony PCR'''. Instead of adding purified ''lsy-2'' gene fragments as template DNA in a PCR reaction with primers specific for your gene (as it was done to make the plasmid), you will add a TINY little part of a colony as the template for your PCR reaction. During the first heat cycle the cells will burst open and release their DNA into the reaction. We will test both well-isolated, non-satellite colonies per group to be sure we continue with bacteria that have the gene of interest inserted in our plasmid.<br>
# Obtain a previously prepared culture of ''E. coli '' bacteria that have been transformed with pPD129.36 lys-2 and growing on solid Luria broth medium with ampicillin. Select two large, well isolated colonies and label them A and B on the bottom of the plastic plate using your fine tipped Sharpie. Label the plate on the bottom with your initials, your lab day, and add a small strip of your team color tape.
#Obtain three PCR tubes and lids from your instructor in your team color.
#Label the side of each pcr tube A, B and C - the marker WILL rub off the top. Tube C will serve as your negative control with no colony added.#Add 30 ul of master mix to each tube. Your master mix will include: 23 μL H<sub>2</sub>O; 3 μL of PCR buffer (10 mM Tris, 50 mM KCl, 1.5 mM MgCl2 pH 8.3); 0.67 μL of 10 mM dNTPs; 0.67 μL of forward primer (20 μM stock); 0.67 μL of reverse primer (20 μM stock); 2 units/μL Taq Polymerasepolymerase
#After each tube has master mix, use the sterile end of an autoclaved toothpick (''not'' the end you are touching) or the end of a sterile micropipet tip and gently touch the center of your colony of interest and pick up a tiny, barely visible amount of the bacteria. DO '''NOT TAKE THE ENTIRE COLONY!!!'''
#Gently twirl the toothpick in the tube or mix the bacteria from the pipet tip with the master mix making SURE that you have gotten it off the tip or toothpick and into the reaction. Discard the toothpick or the tip into your orange autoclave bag.
#Repeat for colony B
#Snap the lid on the tubes, pulse them briefly in the microcentrifuge with the appropriate rotor and adapters adapter and bring them to the thermal cycler for PCR initiation.#Finally wrap , seal the plate in Parafilmwith parafilm. Store the plate Invert it (agar side up) and store it in the refrigerator in your lab day's rack until the day before the next lab when you will set up an overnight culturefrom THIS colony.


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