10. Several lab eroups independently carried out the ligation of a GFP fragment
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10. Several lab eroups independently carried out the ligation of a GFP fragment with EcoRI "sticky-ends" and a vector called pET. pET carries ampicillin resistance and was also cut with EcoRi in the multiple cloning site for E coll were # of Colonies on LB/Amp Plates Material used for transformation in E.coll Group 4 1500 360 Group 3 Group 1 300 850 Group 2 3 ul of ligation reaction using A 529 EcoRI cut pET GFP insert 3 of ligation reaction using EcoRI cut pET only. B 3 l of ligation reaction using 529 EcoRI cut GFP insert alone D131 of uncut pET E No DNA 975 975 975 975 975 a. (1 point) Reactions (A-E) were carried out in paraliel. The results obtained by each of the groups were as reactions were done correctly, which group obtained the most follows. If the ligation and transformation reasonable (expected) results? (Circle one b(1 point) Which of the above transformations (A-E) would you use to determine the transformation efficiency of your competent cells?Explain why in 1 sentence. c. (1 point) How could you explain the results for group 4? d. (1 point) What does the reaction in E control for? e (1 point) What does the reaction in B control for?Explanation / Answer
a. Group 1 obtained most reasonable results.
Reasons:
i. They obtained colonies in the vector + insert tube
ii. They did not get colonies in the insert alone tube
iii. They obtained more number of colonies in the vector alone controls.
b. Transformation efficiency of competent cells is tested using supercoiled DNA. Hence tube D would give the result for transformation efficiency calculation.
c. Group 4 obtained colonies in insert alone control as well. This shows that their experimental set up is contaminated.
d. Reaction E is a negative control. This shows that competent cells do not carry any resistant genes in them.
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