Ligation

newsnet customer via methods%40net.bio.net (by customer from newsnet.com)
Mon Oct 16 18:01:00 EST 2006


"Tom Knight" <tk from mit.edu> wrote in message
news:vuyirike6cn.fsf from shaggy.csail.mit.edu...
> "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.

I was told a 1:3 ratio is preferred because it would increase the chances of
an insert comming into contact with a vector. You can read on some T4 Ligase
information sheet that 1:3 is suggested and 1:1 also, so there is no exact
science.

> 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.

Well I have a 5kb vector and a 1kb insert.
I plan the following:

vector (17ng)
insert (10ng)
10x Ligase buffer
T4 Ligase

Any harm in putting more ligase in than needed? e.g., 1 ul because its easy
to pipette?

Cheers,
ST





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