Double digest: efficiency - what does 50% active in given buffer mean? (Jun/06/2006 )
I am trying to do a HindIII / EcoRI digest and have been using the NEB EcoRI buffer. Someone told me that this was fine, even though HindIII is only 50% active in this buffer, and that as long as you add more HindIII it will still digest fine.
In other words, they said that the 50% activity rating means that 50% of however much enzyme you add will work, and therefore that adding more would overcome the problem. I thought it would mean that only 50% of your template will be cut, and adding more enzyme would not help?.
I have since found another buffer in which they both work, but was just wondering what the 50% activity actually means?
thanks in advance this site rocks!
I think the activity is related to the amount of DNA digested in a given time for a given quantity. For example the unit definition is the amount of enzyme required to digest 1ug of a given DNA in 1 hour. Therefore if something is 50% active it will still digest but only achieve 500ng in 1 hour, therefore if you double the amount then you will achieve complete digestion.
Hope this helps,