T4 RNA Ligase

ChenHA hzhen at freeuk.com
Thu Feb 24 20:33:34 EST 2000


Marieluise Kirchner wrote:

> used anyone the T4 RNA ligase for ssDNA / oligonucleotide-ligation? in
> the buffer of my protocoll hexamine cobalt chloride is used. Why? Is the
> effect really that big? Has hexamine cobalt chloride any bad effects in
> further reactions like PCR or ligation into a vector?
> The question is - should i use this hexamine cobalt chloride or better
> not?

I can't tell you whether you should use cobalt hexamine cobalt or not, but
you should be aware that it works by condensing DNA, and for ligation of
dsDNA, it tends to produce intermolecular ligation products, therefore you
don't get circularised DNA, but long chains of linear DNA.  Quite useless
for normal cloning.  In order to help circularise the DNA, you need K+ in
the buffer.  Personally I don't like to use hexamine cobalt, and I would
leave it out for dsDNA ligation.  However, I have no experience of ligating
ssDNA, so I can't advise you on that.

See
Rusche & Howard-Flanders, Nucleic Acids Res 1985; 13(6):1997-2008


>
> thanks and a nice evening
>
> marie
>
> ---





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