Problems ligating

Frank O. Fackelmayer Frank at Fackelmayer.de
Mon Jun 18 09:36:18 EST 2001


Hi Rhoda,

>   My insert and vector are
> cut,and clean.  I treated the vector with shrimp alkaline phosphatase
> (SAP) and all enzymes have been inactivated according to manufacturer's
> specification.  My controls show that SAP, and T4 ligase are working.  

I have given up using alkaline phosphatase. Although in theory it is
great, it makes more problems than it solves.

> However, when the reactions with insert and vector are examined on the
> gel there is high molecular weight bands not previously present but the
> vector band is still very prominent. 

we never run ligation reactions on a gel, it does not make much sense
because there are too many possible products and you can hardly identify
one particular of them. In addition, we have the best success in cloning
with only very little vector added. When you say you see it as a "very
prominet band", it is way too much. Normally, when you use 10-50ng of
cut vector, you won´t see very much of it on a gel.


> It's intensity is close to the
> amount initially added, so it makes me wonder if the high molecular
> weight bands are just recircularizing inserts.

all DNA with compatible ends will be ligated, giving you a lots and lots
of different products, also in the high molecular weight region. It is
the bugs that select for you, as they do not replicate multiple vectors,
linear products, etc. Therefore, your colonies will have a very limited
repertoire of plasmids, usually only "correct" clones and religated
vector, some others (two or more inserts, especially with small inserts)
and a low percentage of "don´t ask me what it is"-kind of junk.


> Please help I don't know
> where I'm going wrong. I've played with different ratios of insert to
> vector, using 1:1 upto 1:4 and still there are very few white colonies.

unless you are construcing a library, ONE clone is enough! I remember a
difficult cloning with only two colonies on the plate. One was ok, the
other one was vector only. The good one is now the mother of a lot of
our constructs...


> My transform my cell by electroporation and I don't think the problem is
> there.  

check it with uncut, supercoiled vector, but consider that the
efficiency will be way lower for ligation products (100fold minimum).
For difficult cloning, it is important to use the best possible
bacteria. An efficiency of 1x10exp8 per ug should do well.

Frank




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