Digestion Problems

[Tom Hanson]

My guess, and mind you it is just that, is that you are carrying over salt  
or organics from the precipitation or extraction which is inhibiting the 
second digest.  My suggestion would be to do a heat inactivation of the KpnI 
followed by adjustment of buffer conditions and then the Eco digest.  
Alternatively you might be able to find buffer conditions where both enzymes 
are active and do both at the same time.  If you can't find such a condition 
make sure that you are using a goodly volume of 70% EtOH after the precip. to 
wash the pellet and that you vacuum dry to remove any organic carry-over.


Tom