Label one tube with your initials and a (+) and the other tube with your initials and a 2. Transfer 2-4 large colonies using a sterile plastic loop to each microelectronic tube and completely respond. Do not transfer any agar. Put the tip of the loop into the Cacao solution and spin until there is not any cells on the loop. . Close each of the tubes and put them in ice. 4. Ask your teacher to use a P-20 microcomputer to add pogo DNA to your transformation mix. 5. Add pogo DNA to the (+) labeled microelectronic tube. 6.
Incubate both microelectronic tubes on ice for fifteen minutes. 7. Take both tubes out of ice and immediately place in incubator at ICC for 90 seconds. 8. After place both tubes back in the ice for two minutes. 9. Add lull Lurid Recovery Broth to both microelectronic tubes. 10. Let both the tubes rest at room temperature for 10 minutes. 11. During the 10 minutes, get the LB agar and LB+AMP agar plates ready. Mark your plates with the transformation tube mixture to use (+ or -), the lab group names, and the date on the top of the dishes. 12.
Add lull of the pogo transformation cell mixture to the center of the agar surface of the corresponding LB agar and LB+AMP plates. 13. Use a sterile plastic loop to distribute the cell suspension evenly on the plate by “skating” the loop back and forth across the LB agar plate several times. 14. Use the same loop and technique to spread the same cell suspension (+) on the LB+AMP agar plates. Dispose of the sterile loop in a beaker of germicide. 15. Repeat the procedure by spreading the (-) transformation cell mixture to each of the (-) labeled LB and LB+AMP plates.
Be sure to use a fresh plastic loop for the ‘None’ transformation mix. 16. Stack your group’s set of plates on top of one another and tape them together. The plates should be left upright position to allow the cell suspension to be absorbed by the agar. 17. Place the plates in an inverted position (agar side on top) in a ICC bacterial incubation oven for overnight incubation (15-20 hrs. ). Day after lab 1. Lower the lighting in the room and use a long wave I-JAM. Light to visualize he transformed cells that will glow due to the expression of the green or blue fluorescent proteins.
Data: (Positive Control) LB/AMP+ (Experimental) LB/AMP- Bacterial Growth lawn 3 colonies No growth Conclusions: The bacteria treated with the PAM solution developed a resistance to inclining and were able to grow on the inclining plate. Those that were not treated with the PAM were not able to grow on this medium. The plates with no inclining served as a control to show how the bacteria would look in normal conditions. Transformation is never fully effective, Only cells that are competent enough are able to take up the foreign DNA.
Therefore, the inclining* plates showed less growth that the control plate. Questions: 1 . Record your observations about the color and growth (number of colonies) of bacteria on the Petri plates. If you have so much bacterial growth that you can’t count individual colonies, this is referred to as “lawn. ” no growth 2. Calculate the transformation efficiency of your transformation experiments. Transformation efficiency refers to the number of cells transformed per micrograms (jug) of DNA. The transformation efficiency of my transformation experiments is 0. 0125 cells transformed per micrograms (jug) of DNA.