Transposon%2520Mutagenesis%2520Session%25202%2C%2520In-Lab%2520Group%2520Assignment

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Dec 6, 2023

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Transposon Mutagenesis Session 2, In-Lab Group Assignment (10 points) 1. Are the results from plate #1 as predicted in Tn Mutagenesis Session 1? Why or why not? (0.5 pt) The results from plate #1 are what we predicted. There is a lawn of colonies on the plate, which means there was lots of growth, which we predicted. Also, the colonies do not glow, which is what we predicted. 2. Are the results from plate #2 as predicted in Tn Mutagenesis Session 1? Why or why not? (0.5 pt) The results from plate #2 are what we predicted. There was no growth on plate #2, which was as predicted. Also, there were no glowing colonies, which we predicted. Your lab instructor will determine the average number of glowing and non-glowing colonies from the unmutated and mutated pGLO plasmids from your data. Record this data (from your lab instructor) in the table below: plasmid # glowing (green) colonies # non-glowing (white) colonies unmutated pG (C) 1031.75 0 mutated pGLO (M) 36.5 13
3. Remember that transformation efficiency can be calculated by determining the number of colonies resulting from the transformation of 1 ug of plasmid DNA. To measure the efficiency of transformation, not mutagenesis, transformation efficiency should be calculated from the unmutated, control, pGLO plasmid. In Tn Mutagenesis Session 1, you transformed your competent cells with 2 ng of the pGLO plasmid. What was your lab section’s average transformation efficiency? (1 pt) The average number of unmutated pGLO colonies is 1031.75 colonies. 2 ng of pGLO plasmid was used, which means .002 ug of pGLO plasmid was used. The average efficiency is 1031.75 colonies/ .002 ug of DNA = 515875 colonies/ug. 4. For each different color colony (glowing versus non-glowing) obtained on the mutated pGLO (M) plates, where did the transposon likely insert in the pGLO plasmid based on the observed phenotype? (1 pt) For the non-glowing colony obtained on the mutated pGLO plates, the transposon was likely inserted into the coding region of the GFP or araC genes in the pGLO plasmid because these genes control whether or not the cell fluoresces under UV light. For the glowing colony obtained on the mutated pGLO plates, the transposon was likely inserted into any region of the pGLO plasmid except for the araC, GFP, bla, or ori regions of the plasmid. 5. Of the total number of mutated pGLO colonies, what percentage were G- mutant colonies? Briefly explain how you determined this percentage. (1 pt) There were 13 G- colonies out of an average of 49.5 mutated pGLO colonies. This means 13/49.5 *100 = 26.3% of the mutated pGLO colonies were G- mutant colonies. Restriction Digestion Experimental Design 6. (1 pt) Which enzyme(s) would you like to use in your restriction digestion experiment? Make sure to write these enzymes here legibly and without typos. The enzymes we would like to use are NdeI and PsiI. 7. (0.5 pt) Do these enzymes work with 100% activity in the same buffer system? Which buffer system will you be using in your experiment? Briefly explain in 1 sentence why it would be an issue if the enzymes you wanted to use worked at different activity levels in a single buffer system (Hint: think about what would happen if 1 enzyme worked with 100% activity and the other worked with 50% activity with regards to how they would digest the target DNA).
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