1. You are attempting to clone an EcoRI fragment into an EcoRI-restricted vector
ID: 261330 • Letter: 1
Question
1. You are attempting to clone an EcoRI fragment into an EcoRI-restricted vector treated with Calf-intestinal alkaline phosphatase (CIP), and you obtain the following results on your transformation plates (with Amp):
#1 vector only + ligase : 107 colonies
#2 Vector + insert + ligase : 663 colonies
a. Would you expect more, fewer or about the same number of colonies if no ligase were added in #2? Explain in detail, answer should be answered in at least two sentences.
b. Would you expect more, fewer or about the same number of colonies with #1 with a vector not treated with CIP? Explain in detail, answer should be answered in at least two sentences.
HO O ....DNA STRAND OH HO O C H CH 2 calf intestinal phosphatase +H20 5' termni base baseExplanation / Answer
1. (a.) Fewer colonies would be expected in such a case as the vector has been treated with Calf-intestinal phosphatase (CIP) already, thereby losing self-ligation property. Hence, ligase would be required for the annealing of DNA fragments in the absence of insert and thus, to obtain the optimum number of colonies after transformation.
(b.) If the vector isn't treated with CIP, it would tend to self-ligate, hindering for the insert to make a place and ligate efficiently with the vector. Hence, a fewer number of colonies would be expected if the vector isn't treated with CIP.
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