i've just read the fantastic paper of prof Achilles Dugaiczyk (1975)
about "Ligation of EcoRI endonuclease-generated DNA fragments into
linear and circular structures". He tries to foresee the results of a
ligation, known DNA concentration and lenght. The study is centered
on the ligation of a population of one or two kind of fragments, without
too much concern about cloning procedures, that include a ligation to
produce as much as possible mono-eterodimeric recombinants (1 of the
vector, 1 of the insert).
I'd like to undergo some thoughts i've done. The j/i ratio means
something only in respect to a kind of fragment. The value of j has no
meaning for a molecule with different ends, isn't it? Thus the ratio can
be calculated only alter the first reaction of a Ligase over 2 different
DNA molecules, which gives a fragment (the linear version of what will
have to enter bacteria) with complementary ends.
This first reaction should procede with a DNA molar ratio of 1:1,
because i want a mono-eterodimeric recombinant, right? Thus the molar
ratio of the fragments to be ligated shouldn't be changed too much
(particularly when recombinant is >10kb, as some calculations show) to
raise the probability of selfcircularization (of this preligated
molecule), otherwise the j/i ratio will drop under the unit: j become
lower because there are more probably many preligated fragments with 3
or 5 inserts (DNA longer, j lower).
However with the progression of the reaction i gets lower too (less free
ends) and the wanted recombinant starts to accumulate.
Any comments are #really# welcome!
Thanks, best regards