Double restriction digest

Karl Fischer tyr-2 at bones.biochem.ualberta.ca
Sun Dec 14 23:22:36 EST 1997


In article <19971215014701.UAA22247 at ladder02.news.aol.com>,
rick00100 at aol.com (Rick00100) wrote:

<snip>
>So my question is:  how do you make sure the vector is cut completely by
2 >enzymes? 

Hi Rick,

Two questions
Question : does the double-cut vector give a good deal of colonies when
self-ligated and transformed?
Question : how close are the two sites?

On the first point - if the self-ligation test shows a good deal of
colonies then you do have poor second cutting (I'm going on the assumption
you gel purify the linearized vector after the first cut).

As to the second quesiton on page R56 of the Gibco/LifeTech catalogue
(usual disclaimer) they give the digestion efficiency of double digestion
of pUC19 MCS. Bottom line - second digestion with some enzymes are partial
or downright negative depending on how close they are to each other.

One possible fix is to phosphatase treat the linear vector - any single
cut species should, in theory, not be able to religate. A handy trick is
to dephosphorylate the vector following first digestion and prior to a gel
purification - following this a quick self-ligation/transformation will
give you an idea of the maximum background you can expect if, in fact, the
second RE digest is unsuccessful or only partial.

Just some Sunday night musings,

Karl the hepB guy



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