pGlo Lab

Mrs Muñiz's Bio is Beautiful

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pGlo Lab

1. Label one closed micro test tube +pGLO and another -pGLO. Label both tubes with your group's name. Place them in the foam tube rack.

2. Open the tubes and, using a sterile transfer pipette, transfer 250 µl (microliters) of transformation solution (CaCl2) into each test tube.

3. Place the tubes on ice (beaker with ice and water)

4. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Pick up the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the –PGLO tube.

5. Examine the pGLO DNA with the UV lamp. Note your observations. Immerse a new sterile loop into the pGLO plasmid DNA stock tube. Withdraw a loopful. There should be a film of plasmid solution across the ring. This is similar to seeing a soapy film across the ring. Mix the loopful into the cell suspension of the +pGLO tube. Close the tube and return it to the rack on ice. Also close the –pGLO tube. DO NOT add plasmid DNA to the –pGLO tube.

6. Incubate the test tubes on ice 4 for 10 minutes. Make sure bottom of tube makes contact with the ice.

7. While the tubes are sitting on ice, label your Four LB Nutrient agar plates on the bottom (not the Lid) as follows:

· Label one LB/amp plate: + pGLO

· Label the LB/amp/ara plate: + pGLO

· Label the other LB/amp plate: - pGLO

· Label the LB plate: -pGLO

8. Heat shock. Using the foam rack as a holder, transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42°C, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.

When the 50 seconds are done, place both tubes back on ice. For the best transformation results, the transfer from the ice (0°C) to 42°C and then back to the ice must be rapid.

Incubate tubes on ice for 2 minutes

9. 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 250 µl of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 minutes at room temperature.

10. Tap the closed tubes with your finger to mix. Using a new sterile pipet for each tube, pipet 100 µl of the transformation and control suspensions onto the appropriate nutrient agar plates.

11. Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the LB nutrient agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface. DO NOT PRESS TOO DEEP INTO THE AGAR.

12. Stack up your plates and tape them together. Put your group name and class period on the bottom of the stack and place the stack of plates upside down in the 37°C incubator until the next day.

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