Blunt end ligation debate. >>GEL PURIFY YOUR VECTOR
Dennis J. Templeton
djt2 at po.CWRU.Edu
Fri Jul 16 12:31:40 EST 1993
In a previous article, JANIEMI at FINABO.ABO.FI (Jarmo Niemi) says:
>In <1993Jul14.085421.25969 at aristo.tau.ac.il> pc386 at ccsg.tau.ac.il writes:
>> Following transformation, it was clearly evident that the CIP reaction failed
>> to perform, as I obtained many colonies in the control plate of Blunted/CIPped
>> self-ligated vector.
>It has been my impression that CIP not working is a sadly frequent
>problem, which is often not easy to solve. It appears that there
>still are bad batches of CIP from even the most reputable
>manufacturers, so try another batch or another manufacturer. If
>you find a good batch, HOARD it...
Here's my 2cents
Many people who tell me that their cip doesn't work say that because their
cipped vector prep gave "background" colonies. In almost all of these
cases the complainant did not gel purify their vector. Given that, they
cannot be sure if the failed step was the cip, or an incomplete digest of
the starting vector. It is a commonly observed phenomenon that plasmids
isolated by denaturing steps (i.e. boiling or alkali) *always* have a
minute amount of permanently denatured DNA that will not cut. You will
never get rid of this plasmid with restriction enzyme or cip, but will with
a simple gel purification.
I teach my folks to *always* gel purify their vector. After all, you are
usually purifying the insert, not? It's just one extra lane on the gel.
We cip treat with 1 u of Boehringer cip (dated 1986) per ug of vector,
right in the restriction buffer for 20 minutes after digestion, then load
the whole reaction on an agarose gel and glass powder purify the linear
Typically, a 50 ng reaction of 5' overhangs gives no background colonies,
but about 50-500 with insert. With blunts, it's a little tougher, about 5
background and 30-50 with insert. With 3' overhangs, it's hardest with
usually 50 or so background and if you're lucky 100 or more with insert.
For 3' overhangs, we usually either force clone them (with 2 different
restriction ends) or do the cip reaction (still in restriction buffer,
though the purist might add cip buffer, pH 9 Tris + 1mM Zn++) at 55
degreesC with more cip.
But I emphasize... gel purify your vector. Trust me.
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