Triple restriction digest?

Sat Oct 14 03:44:01 EST 2000


Run a longer agarose gel (may be 24cm) for a longer time.
You should be able to separate them quite easily.
3.5 and 2.5 kb should get separated in a normal sized (12cm) 0.8% agarose
gel also, if you run them for a longer time.

Hope this helps.

On Fri, 13 Oct 2000 mbraden23 at wrote:

> 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
> Sent via
> Before you buy.


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