Question

Part 2: Complete Ligation Reaction Calculations

You are setting up a ligation reaction with the following components:

Plasmid Vector (0.02 µg/µL)

Insert DNA (0.03 µg/µL)

10X T4 Ligase Buffer

T4 DNA Ligase Enzyme

Water

You plan to set up one experiment with a _______ ratio of insert to plasmid. The amount of plasmid vector to be used in total will be _____ µg. The final reaction volume will be 50 µL. You will add 2.5 µL of T4 Ligase once the reaction samples are prepared (for a total volume of 52.5 µL). Complete the following chart regarding the volumes of each component used to prepare your samples.

Ratio	Volume of Plasmid Vector (0.02 µg/µL)	Volume of Insert DNA (0.03 µg/µL)	Volume of 10X T4 Ligase Buffer	Volume of Water	Total Volume
					50 µL plus 2.5 µL of ligase

Answer 1

Well first of all the calculation may change for the different insert sizes.

You may use any ratio of the plasmid to insert from 1:1, 1:2, 1:3 and so on. I will give an example of 1 kb insert size and 10 kb plasmid DNA for a 1:1 ratio. For any ration, we keep vector concentration constant

1) for 1:1

2) As vector concentration is constant, 1ul or 0.02 ug of the vector is required in 1:1 ratio with the and insert concentration of 2ng.

Again for further example for 1:5 ratio, keeping plasmid concentration of 0.02 ug the insert required will be 10ng.

I would prefer to use more insert to plasmid ratio to increase the chance of ligation.