Question

To obtain 500 ng of a 3 kb fragment how much amount of plasmid needs to be digested

Answer 1

8333 microgram vector.

For a digest reaction, one can scale up to 100 microL. Keep the glycerol concentration at 5% or less.

Almost all restriction enzymes have 50% glycerol. Hence, the enzyme volume must be 10% or below. For many enzymes, 1 microL is sufficient for 1 microgram of DNA. Before starting a reaction, plan for loss of material at the time of gel purification.

If two microgram vector is needed, start with double of that. For an insert, a 180 bp insert comes to 180/3000 = 0.06. This is the mass of the intact plasmid.

Hence, for 500 ng vector, one may need 500/0.06 = 8333 microgram vector.