Question

8. You have two plasmid preparations A and B (illustrated below) each at concentrations of 100nguL. You wish to subclone a 1.5 Kbp EcoRI fragment from plasmid·A into the unique EcoRI site of a 3.0Kbp vectorB. EcoRI 1.5 Kbp Total size 3.0Kbp BT Total size 7.5Kbp Complete the following table to indicate the volumes of each ingredient required for the preparation of a 50μL ligation mixture containing 50ng of vector and an amount of digested plasmid A required to have an insert to vector ratio of 3:1. Ingredient Volume (μL) EcoRI digested plasmid 50ng EcoRI digested Plasmid B* 5X ligation buffer T4 DNA ligase Water 10

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Answer #1
Ingredient Volume (uL)
EcoRI digested plamsid A 1.5
50ng EcoRI digested plasmid B 0.5
5X ligation buffer 10
T4 DNA ligase 1
Water 37

Plasmid A stock concentartion is 100ng/ul we need 150ng so 1.5ul

Plasmid B stock concentration is 100ng/ul we need 50ng so 0.5ul

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