In this lab, we will obtain a better understanding of bacterial transformations using pVIB. Hypothesis: If the bacteria transforms successfully with the pVIB, the the +plasmid (LB & AMP) will glow in the dark. III. MATERIALS & PROCEDURES 1. Use one sterile 15-mL tube “-plasmid;” and another “+plasmid. ” 2. Use a sterile transfer pipet to add 250 ? L of ice-cold calcium chloride each tube. 3. Place both tubes into ice. 4. Use a sterile plastic inoculating loop to transfer one large colony of E. coli cells from the starter plate to the +plasmid tube. . Return +plasmid tube to ice. Transfer a second mass of cells to -plasmid and do the same as +plasmid and mix thoroughly. 6. Both tubes should now be on ice. 7. Use the sterile plastic inoculating loop to transfer one loop full (10 ? L) of pBLU solution (0. 005 ? g/? L) to the +plasmid tube. Lift the loop full of pBLU solution into cell suspension, and mix DNA. 8. Return +plasmid tube to ice, and incubate both tubes on ice for 15 minutes. While tubes are incubating, label 2 LB plate and 2 LB/Amp plates with lab group name and date. a) Label one LB “+”. Experimental plate).
b) Label the other LB “-“. (Negative control).
c) Label one LB/Amp plate “+”. (Experimental plate).
d) Label the other LB/Amp plate “-“. (Negative control).
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9. A 15-minute incubation is followed by a “heat shock”. Remove both tubes directly from ice and immediately immerse them in the 42° C water bath for 90 seconds. Return both tubes directly to ice for one minute. 10. Use the sterile transfer pipet to add 250 ? L Luria Broth to each tube. Gently mix, and set the tubes in a test-tube rack at room temperature for a 10 minute recovery. 11.
Use the sterile transfer pipet to add 100 µL cell suspension from –plasmid tube onto each appropriate plate. Repeat for the +plasmid plates. 12. Spread the cells over surface of the “-” plates. Use sterile transfer pipet to add 100 µL cell suspension from +plasmid tube onto the +LB/Amp plate. 14. Allow the plates to set for several minutes, then wrap with tape. Place the plates upside down in 30° C incubator, and incubate for 36 to 60 hours. Ask teacher to incubate your plates. You should have FOUR in total. IV. RESULTS/DATA COLLECTION/ANALYSIS | +LB| -LB| +LB +AMP| -LB +AMP|
Type| colonies| colonies| colonies| none| size| 3/4 tray, colonies about 1mm in size| whole slide| some about 1mm in size| no growth| #| 3-4 colonies| 3-4 colonies| around 11 large colonies and 36 small| none| color| yellow-white| yellow-white| yellow-white| clear| glow| NO| NO| YES| NO| V. DISCUSSION/CONCLUSION The expected results were confirmed. There were no restrictions in +/-LB plates, there was just growth as the E. coli bacteria reproduced. However, as the ampicillin was introduced in the +/-LB/Amp there were changes.
In the –LB/Amp there was no growth since the bacteria had no resistance towards the antibiotic, but in the +LB/Amp there were colony growth from the bacteria, showing resistance. In the dark, the +LB/AMP plate glows, showing a fluorescent effect. There could have been multiple errors. I believe that the timing could have been slighty off, causing a differentiation in the bacterial growth, possibly even nullifying it. I didn’t put enough Luria Broth into the plates, subsequently giving us less colonies since there aren’t enough nutrients.
... from other bacteria plasmids, or even eukaryotes into recipient bacterial cells. Materials and Procedures I marked one sterile 15-mL tube + and ... there were no plasmids to resist the ampicillin in the LB/amp- plate, there was no growth. Introduction The bacterium used in this ... of a pencil eraser from isolated colonies of E. coli cells from the starter plate into the + tube. I immersed these cells on ...
Next time, we will try our best to get accurate measurements of the solutions, especially the Luria Broth. VI. LITERATURE CITATION Campbell, N, & Reece, J (2005).
Biology 7th edition, AP. San Francisco, CA: Pearson, Education Inc.. The AP Biology Development Committee, (2001).
Biology lab manual. New York, New York: The College Examination Board. VII. QUESTIONS 1. What are you selecting for in this experiment? We are selecting for transformed bacteria that glows in the dark. 2. What does the phenotypes of the transformed colonies tell you?
The phenotype tells me that the plasmid successfully transformed within the bacteria. 3. What one plate would you first inspect to conclude that the transformation occurred successfully? Why? I would choose the +plasmid (LB/AMP) plate. This plate proves my hypothesis and is critical for evidence as part of a successful experiment. 4. What factors might influence transformation efficiency? Explain the effect of each factor you mention. The Luria Broth, nutrient medium, would help influence transformation efficiency. This is due in part because the bacteria need nutrients in order to reproduce.