Triple restriction digest?
SURESH KUMAR K.G.
skg at biochem.iisc.ernet.in
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 my-deja.com wrote:
> 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 Deja.com http://www.deja.com/
> Before you buy.
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