Help with Enzymatic Calculations

Dr. Duncan Clark Duncan at genesys.demon.co.uk
Sat Jun 5 10:18:55 EST 1993


In article <1993Jun5.060506.6712 at news.weeg.uiowa.edu> rcuevas at news.weeg.uiowa.edu writes:

> 
> 
>I hope somebody can help me out with this (potentially easy) problem.  :)
>I'm currently a second year biology student, and I'm gradually learning the
>laboratory techniques involved with molecular biology (ie. electrophoresis,
>blotting, uses of enzymes/buffer/DNA solutions, etc.)  Right now, I'm learning
>the methods involved with restriction enzyme digestion, and I must admit that
>I'm having a difficult time understanding how to determine the "correct"
>amount of restriction enzyme to use for a given amount of DNA sample - to be
>more specific, I don't understand how the concept of "units" fits into the
>overall schema (I haven't had any formal classes in molecular biology or
>genetics, so I don't know if this is an "elementary"-type of question.) For
>example, given that I have 500ng=0.5ug of DNA diluted in 20uL of solution, and
>also given that the enzyme (stock) solution contains 20,000 "units" per mL,
>how do I determine the volume amount of enzyme solution to use (based on the
>"units" concept).  Any help is most greatly appreciated. Thanks!
>
>
>Ramon F. Cuevas
>rcuevas at umaxc.weeg.uiowa.edu
> 
> 
>
>
1 unit of a restriction enzye is usually defined as the amount of enzyme
that will cut 1ug of (usually) lambda phage DNA in the appropriate buffer at the
appropriate temperature in a volume of 50ul in one hour.

That definition is true for most 'clean' linear DNA's. If you are cutting
supercoiled plasmid DNA then usually more enzyme is needed. For Xsomal DNA 
again more enzyme is usually needed. 

In your case add say 5-10u max. ie 0.5ul. It doesn't matter if you use too much 
providing you don't do anything silly ie 200u with 0.5ug DNA  = 400fold 
overdigest. Most enzymes will easily allow 100fold but beware anything more 
regardless of what the data sheet says. Obvioulsy the more you use the more 
expensive it is so for expensive enzymes use less enzyme and increase the 
digest time ie several hours to overnight.


-- 
Dr. Duncan Clark
Enzyme producer 
Duncan at genesys@demon.co.uk



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