Triple restriction digest?

mbraden23 at my-deja.com mbraden23 at my-deja.com
Fri Oct 13 17:32:17 EST 2000


Hello,

I was wondering if it is uncommon or not reccomended to do triple
restriction enzyme digests (simultaneously).  I am attempting to
subclone a lacZ gene using a double digest and am having some trouble
separating a 3.5 and 2.5 kb band on the agarose (0.8%) gel for gel
purification.  I believe spreading the digest over more wells on the gel
might give better resolution (from weaker bands), but thought that maybe
if I make even more of a difference in size, it would be even easier.  I
have another restriction site that is compatible with the buffer
conditions and temperature that would cleave off another 0.5 kb or so
off the undesired  segment.  Is this not a good idea/waste of enzyme?  I
have another double digest protocol, however it requires two enzymes
that are not compatible, and would just be a little more work (and would
give the 3.5 and 2 kb that the triple digest would do).  Any advice
would be greatly appreciated.

Thanks and aloha
Mike Braden


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