In this lab we did a procedure known genetic transformation. A gene is a piece of DNA which provides instructions for making a protein. This protein gives an organism a particular trait. Genetic transformation means change caused by genes, and involves the insertion of a gene into an organism.
Materials
- E coli starter plate
- Poured agar plates
- Transformation solution
- LB nutrient broth
- Inoculation Loops
- Pipets
- Foam microtube tube/float
- Container full of crushed ice
- Marking pen
- Rehydrated pGLO plasmid
- 42 degree Celsius water bath
- 37 degree Celsius incubator
Procedures
1. First we labeled the micro test tubes +pGLO and -pGLO
2. Then we added 250 micro liters of transformation solution(CaCl2) using the pipet into each tube.
3. Next we placed the tubes inside the foam float and put it in ice for 10 minutes
4. Using a sterile loop, we picked up a single colony of bacteria from our starter plate. We put a single colony of bacteria in both the +pGLO and -pGLO tubes.
5. Then we put a sterile loop inside into the pGLO plasmid DNA stock tube. Once we withdrew a loopful, we only placed it in the +pGLO tube.
6. Next we incubated the tubes on ice for 10 minutes more.
7. Label the four LB nutrient agar plates (LB/amp +pGLO) (LB/amp/ara +pGLO) (LB/amp -pGLO) (LB -pGLO)
Hypothesis
- The +pGLO LB/amp will have normal amount of bacterial growth
- The +pGLO LB/amp/ara will have bacterial growth and glow under UV light
- The -pGLO LB/amp will have no bacterial growth
- The -pGLO LB will have a lot of bacterial growth
8. Using the foam rack as a holder, we transferred both the +pGLO and -pGLO tubes into the water bath, set at 42 degrees Celsius for exactly 50 seconds. After the 50 seconds we then placed the float back on ice for 2 minutes.
*needs to be rapid transfer
9. After the 2 minutes we used a pipet and added 250 micro liters of LB nutrient broth to both tubes and reclosed it.
10. Mix the LB broth and solution in the tubes. Then using a pipet for each tube, pipet 100 micro liters of the transformation solution and control suspensions onto the appropriate nutrient agar plates.
11. Using a sterile loop for each plate, we 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 fourth across the surface of the plate.
12. Last, we stacked our plates and taped them together. We put them inside an incubator at 37 degrees Celsius until the next day.
Results
LB/amp +pGLO
LB/amp/ara +pGLO
This top photo shows a better transformation efficiency because more colonies of bacteria are lit up.
LB/amp -pGLO
LB -pGLO
Conclusion
After looking at our results, we knew we had done something wrong. Most of our data was correct except for one plate. We believed that we may have switched some solutions around or maybe it was just a flaw in the long, careful steps. The results would be completely screwed over with one tiny flaw in the experiment. Our biggest problem was during the transfer part. We did not transfer the foam float with the tubes in them quick enough which resulted in a little amount of glow in the dark colonies. The next flaw was in the LB amp -pGLO. We expected for bacteria to not grow on the plate, but there was a lot on it. We think that some bacteria contaminated either the tools we used or possibly we used either the same piper or loop twice. With these many ideas I believe that we used the same tool twice. It is the only way I think bacteria could have gotten in the plate. All in all, this experiment was a fun, careful, and interesting lab.
I enjoyed the examining the plates at the end of the experiment the most. I enjoyed this because I could see the results and to see if we completed the procedures correctly. My most valuable takeaway from this experiment would probably be patience. Throughout the experiment we had to wait a lot of the time to finish the steps. Also the preparation that Mr. Wong did to set up the experiment must have taken a while because of the exact measurements and conditions. We could have definitely improved our transformation effiency. We were probably not the fastest group to transfer our foam float from ice to water and back. The better transformation effiency we had would result in how much the colonies glowed. Also with some confusion in the procedures that also slowed the efficiency. We definitely had flaws in our procedures because the LB/amp -pGLO should not have had bacteria on it. Our transformation efficiency was slow, and possibly the measurements and timing were a little off. We could have also mixed up the plates but that is very unlikely. One way to alter the experiment would to use other types of bacteria. Of course different outcomes would occur.
