Re: what are the various factors that can affect the activity of restriction en

There are various factors that can affect the activity of restriction enzymes.  The quality of your 
DNA preparation really has less to do with the amount of protein contamination than with salt 
contamination.  If you are using DNA from a prep kit with purification columns, that is usually 
best; however, if you have DNA from a 'homemade' prep, you will want to centrifuge your DNA 
and resuspend the pellet in new buffer several times to wash out any residual salts.  Other 
factors that can affect the enzymatic activity include:  detergents, alcohol, phenol, or EDTA.  If 
your DNA prep includes any of these agents, perform several more washes of your DNA as 
suggested above. 

Also, a good policy is always to add the enzyme to your sample tube LAST.  Adding restriction 
enzymes to water alone can (and will) denature your enzyme.  Make sure the enzymes are kept 
on ice at all times while in use, and check the manufacturer's expiration date.  If your enzyme 
has undergone multiple freeze/thaw cycles, its activity can be severely reduced, and even though 
the manufacturers build in plenty of time beyond the expiration date, if your enzyme is one that 
gets a lot of use and its date is expired, you may just need a fresh batch.

Finally, if you wish to add more proteinase K than required, it shouldn't hinder your enzyme's 
activity.  However, if you really want to learn more about optimal conditions for your specific 
restriction enzyme, you could check the manufacturer's website and click on the link for 
technical documents.  You may be able to pull up some information that is more relevant to your 
specific conditions.  

Hope that helps . . . .

Sue Baker,
Mad Scientist