Place the tubes on crushed ice. Do not use cubed ice. Examine the pGLO plasmid D
ID: 165496 • Letter: P
Question
Place the tubes on crushed ice. Do not use cubed ice. Examine the pGLO plasmid DMA solution with the UV lamp. Note your observations. Pipet 10uL of pGLO plasmid into the +pGLO tube and mix. Close the +pGLO tube and return it to the rack on ice. Do not add plasmid DNA to the -pGLO tube. Why not? Close the -pGLO tube and return it to the rack on ice. Incubate the tube on ice for 30min. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contac with the ice. While the tubes are sitting on ice, label your four agar plates on the bottom (not the lid) as shown the diagram. Heat shock. Using the rack as a holder, transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42 degree C, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack to the bottom of the tubes stick out and make contact with the warm water. When the 50 seconds have passed, place both tubes back on ice. For the best transformation results, the change from the ice (0 degree C) to 42 degree C and then back to the ice must be rapid. Incubate tubes on ice for 2 min. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250uL of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for -10min at room temperature (optional).Explanation / Answer
4. -pGLO tube is your negative control. a control which ensures that whatever results that you have obtained are due to plasmid only, not due to background.
typically, you should not get any colonies in the -pGLO plate. this will confirm that the colonies that are formed are due to the transformation of the cells with the plasmid, and not due to some backgroung contaminating bacteria etc.,