inserts and vector ratio in liagtion Rxns - (Feb/17/2006 )

Hi ladies and gentlemen:

I wonder how to determine the ratio of a big insert (4.3kb)and clone vector (e.g.pGEM-T easy from Promega?the provider just offer a ambiguous guide as below:

"The pGEM®-T and pGEM®-T Easy Vector Systems have been optimized using
a 1:1 molar ratio of the Control Insert DNA to the vectors. However, ratios of
8:1 to 1:8 have been used successfully. If initial experiments with your PCR
product are suboptimal, ratio optimization may be necessary. Ratios from 3:1
to 1:3 provide good initial parameters".

But according to your bench experiences, what molar ratio is best between such a long insert and the vector?

Many thanks!

I have never understood how the optimal ratio could be anything except a 1:1 molar ratio. The ligase does not know which fragment is the vector and which is the insert. It may be useful to vary the ratios to account for DNA damage, contamination, or measurement inaccuracy for one of the components, but I see no reason to choose one or the other.

If the vector cannot close on itself, and vector-vector ligations cannot occur, I suppose it doesn't matter. But what you're doing by providing more insert than vector is increasing the chance of a vector end and an insert end of coming together for the ligase to act on. Of course, you're also increasing the chance of insert-insert ligations, but these are not replicons, and so won't transform. At initial state, there is much more chance of a vector end to be ligated to a single insert fragment (because single inserts will greatly outnumber insert-insert dimers, and single inserts greatly outnumber linear vector, favoring (from the vector's point of view) linear vector-single insert combinations).

That said, I never even bother with calculating insert to vector ratios; I just try to provide more insert than vector, estimated by their appearance on a gel (I always have a gel of the vector and of the insert, because I always gel purify my digests).

The historical ratio was always 3:1 (insert:vector), but it's just not that critical. This is evident by Promega's own description: if the ratio can vary from 8:1 to 1:8 and one can still anticipate success, how critical can the ratio be? That's a pretty big margin of error...

This is another one of those things (like A260/A280 ratios) that for most routine experiments, people get too hung up on, IMHO...

For a 1.2 Kb fragment to be cloned into pGEM I was using a ratio of 1:3 considering that the insert is 3 times smaller than the pGEM.
for a 4.3 kb I probably would use 1 to 1 since more or less they are the same size.
This ratio things are really annoying as now am tryi ng to clone a 100 bp fragment into a 10 Kb vector.. this means a ratio of 1:100 according to what I was thought.. which I dont think is working

That clear at least my doubts, thanks homebrew

Hi phage 434 and homebrew:

Thanks for your help! and your guide is very appreciated!

Sincerely

Best regards

I feel the same way about this topic as homebrew.

I also purify my digests on a gel before ligations and I find that it is hardly essential to determine an exact molar ratio.

-Matt