(by tk from mit.edu)
Mon Oct 16 08:30:00 EST 2006
"newsnet customer" <customer from newsnet.com> writes:
> Sambrook recommends DNA as low as 10ng for ligation. The low
> concentration is required for DNA to circularise. I was just
> wondering if this concentration is for the vector or insert or both?
> I read on a website that the vector should be 100ng and the insert
> should be anything from 10ng to 100ng (depending on the molar ratio
> and vector to insert size).
> The site also recommends trying different insert concentrations, to
> find out what works best.
> vector 100ng + 10ng insert
> vector 100ng + 20ng insert
> vector 100ng + 50ng insert
> vector 100ng + 100ng insert
> It seems like the vector concentration is fixed at 100ng, while the
> insert can vary from 10ng. What are peoples thought?
> Here is the website: http://www.soton.ac.uk/~kpa/molecol/clone.html
You can read almost anything "on a website." Some, perhaps most of it
might even be right. The problem here is that Sambrook also says that
the vector:insert molar ratio should be 1:1, and the web site is
speaking in nanograms, not picomoles. Indeed, low concentrations of
vector favor recircularization over concatenation. Further, if you
look at the transformation curve of DNA vs. transformed cells, you
discover that high concentrations of DNA do little to enhance the
number of transformants. 100 pg of pUC19 is almost as effective as 1
ng. The vector:insert molar ratio should be 1:1 because the ligase
does not distinguish between insert and vector -- how can it? And
thus there is no reason to favor one or the other in terms of molar
concentration. This can be wrong if there is an underlying bias in
the quality of one or the other. If you know, for example, that only
20% of your insert is cut properly, then what counts is the molar
ratio of the correct fragments. But remember that this applies to the
vector as well.
Almost all ligation failures are really failures in preparation of the
DNA, not in ligation, and not in calculating or varying vector:insert
ratios. The ligation reaction is remarkably forgiving, especially for
cohesive ends. If you are having trouble, look at the restriction
digests, the treatment of the DNA following restriction, the
purification of DNA following PCR, the overhangs of the cut site on
PCR primer addtion of cut sites, UV damage to DNA during gel
excision, and only rarely at the ligation itself.
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