Amt of restriction enzyme - (Nov/02/2007 )
I just noticed an advertisement by fermentas quick-digest enzymes. They claim 1µl of enzyme mix cuts 1µg of DNA.
I was wondering if such rough equation applies to routinely used restriction enzymes as well. I usually use 20units of Fermentas restriction enzymes for cutting about 3µg of DNA. I would like to know what others use.
Thanks in advance.
It would be dependent on the enzyme but most times, the company will tell you that 1U will digest 1 µg DNA in 50 µl volume. I standardly use this ratio when digested DNA and it works.
I normally add the enzyme such that in total it should not exceed 10% of the total reaction volume.
It always works for me.
Depending on how much DNA you got and how concentrated your enzyme is, just adding an amount in order not to exceed 10% of the reation volume seems like a waste of enzyme. Mostly they come at 5-20 Units/µl which should be enough to cut 5-20 µg of DNA...
Yes, most enzymes will cut in 5 minutes at that concentration. Have a look at this: