Question

You are preparing to clone a DNA fragment into a plasmid vector. You start by linearizing your plasmid (concentration = 500 ng/μl) with EcoRI, which is provided in a standard 50% glycerol solution at 10 units/μl. Your enzyme also comes with an appropriate 10X reaction buffer.  Taking into account the final allowable glycerol concentration, you want to add the maximum amount of EcoRI, to achieve complete digestion of 1 μg plasmid DNA in a total volume of 20 μl. Fill in the table below to show how much of each component you would add.

Reaction component
Volume (μl)

Plasmid DNA
EcoRI
10X reaction buffer
Molecular water

When your digest is done, you add 6X Gel Loading Buffer to the reaction and then run it on a 0.9% agarose gel. Determine 1) the amount of 6X GLB to add, and 2) the preparation of 40 ml of 0.9% agarose.

Answer 1

1 μg plasmid DNA in a total volume of 20 μl.

1µg = 1000ng

1µl has 500ng of plasmid

Hence 2µl has 1000ng or 1µg of plasmid

Plasmid DNA = 2ul

Maximum amount of restriction enzyme added to the reaction is 10% of the total reaction volume to get 5% glycerol in final volume

Reaction volume = 20µl

Volume of EcoRI = 10% of 20µl = 2µl

Initial concentration of reaction buffer (C1) = 10X

Final concentration of reaction buffer (C2)= 1X

Final volume of reaction (V2)= 20µl

Volume of 10X buffer needed is V1

V1C1 = V2C2

V1 x 10X = 20µl x 1X

V1 = (20µl x 1X) / 10X

V1 = 2µl

Answer

Plasmid DNA = 2µl

EcoRI = 2µl

10X reaction buffer = 2µl

Molecular water = 14µl