You now set up a ligation reaction with the following components: • Plasmid Vector (0.05 µg/µL)...

You now set up a ligation reaction with the following components: • Plasmid Vector (0.05 µg/µL) • Insert DNA (0.2 µg/µL) • 10X Ligase Buffer • Water Because you are unsure what the best ratio of insert to plasmid is, as this is a new experiment for you, you plan to set up two experiments, one with a 3:1 ratio of insert to plasmid, and one with a 6:1 ratio of insert to plasmid. In both cases the amount of plasmid vector to be used in total will be 0.1 µg. The final reaction volume will be 20 µL. The final concentration of the 10X ligase buffer should be 1X. Using this information, complete the following chart regarding the volumes of each component used to prepare your samples. (Hint: For the plasmid and insert, determine the mass of DNA that needs to be in each tube using the information given, then use the equation C=mass/V to solve for the volume.) Ratio Volume of 0.05 µg/µL Plasmid Vector (µL) Volume of 0.2 µg/µL Insert DNA (µL) Volume of 10X Ligase Buffer (µL) Volume of Water (µL) Total Volume 3:1 20 µL 6:1 20 µL.

Expert Solution

Given that we need to use 0.1ug of Plasmid vector. The concentration of DNA in the stock solution is 0.05ug/ul.

So the volume of plasmid vector = 0.1/0.05 - 2ul.

Given that concentration of Insert DNA (0.2 µg/µL).

Volume for 3:1 ratio - 3:1 ratio means the amount of insert is 3 times of plasmid vector.

Amount of plasmid vector = 0.1ug.

So the amount of insert = 3*0.1 = 0.3ug.

The volume of insert = Amount of insert/concentration of insert = 0.3/0.2 = 1.5ul.

Volume for 6:1 ratio - 6:1 ratio means the amount of insert is 6 times of plasmid vector.

Amount of plasmid vector = 0.1ug.

So the amount of insert = 6*0.1 = 0.6ug.

The volume of insert = Amount of insert/concentration of insert = 0.6/0.2 = 3 ul.

	3: 1 ratio	6:1 ratio
Insert DNA (µL)	1.5	3
Volume of 10X Ligase Buffer (µL)	2	2
Volume of Water (µL)	13.5	12
Volume of 0.05 µg/µL Plasmid Vector (µL)	2	2
T4 DNA ligase (µL)	1	1
Total volume (µL)	20	20

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gladiator answered 2 years ago

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