Question

Create a protocol for a restriction digest with a total volume of 30 µl to digest 1.0 µg of plasmid DNA to completion. You have a restriction enzyme in a standard 50% glycerol at 5 Units / µl but wish to do a standard 'overdigestion' in which you will put the maximum allowable enzyme in the digest. (Indicate how many Units will be in the digest.) You have a compatible 10X restriction buffer, and your DNA is at 0.3 µg / µl. Indicate the volumes of each reagent, including water, if needed, to a final total volume of 20 µl. Indicate as appropriate an order of addition for the reagents.

Final reaction volume must be 20 microliters.

Answer 1

DNA concentration is 0.3 µg/ µl Therefore, We need to take 3.3 µl of DNA from the stock solution to set the restriction digestion.

Buffer concentration to set the Restriction digestion is 1X.

Therefore, we need to take 2 µl of buffer in a reaction volume of 20 µl.\

We need to add enzyme in excess because we need to over digest the DNA. 5 units of an enzyme would be sufficient to over digest the DNA

DNA (1 µg) = 3.3 µl

Buffer      =   2 µl

Enzyme   = 1 µl

H2O    =      13.7 µl\

total reaction volume 20 ul