Just an idea, but since the "stable" transformants are usually the
result of large extrachromosomal arrays, perhaps you could help
the worms by pre-ligating your fragments together, and/or ligating
them into plasmid DNA homologous to "pROL6". If the cosmids that
you say make it work better are partially homologous to the pROL6
vector, then that could be explained. In that case, the worm can
assemble the large array by recombination; whereas that won't
happen with simple restriction fragments.
TRITECH_RESEARCH at LAMG.COM
Phil Morgan, M.D. wrote:
An open question to anyone microinjecting these days.
We've been having pretty good luck with our mutant rescue as of
late, but have one question for the group mind. We have been
rescuing using the rol-6 plasmid marker. When using cosmids
as the coinjected DNA we get about a 20% incidence of stably
inherited lines in the F2 generation and a very good incidence
of F1 rollers (though I don't have the numbers in front of me).
When we change and use fragments of DNA as the coinjected DNA
to search for rescue, we again get a very good incidence of rollers
in the F1 generation, about the same as when using cosmids.
However the incidence of stable roller lines is much less than
seen with cosmids or with no coinjected DNA, about 0.2 to 1%. We
still get rescue of the mutant phenotype, but obviously have to
score a lot more F1 rollers to get enough F2 lines. We have tried
the usual tricks from cosmid injections (running the concentrations
up and down), but so far no effect. Our preparations of fragments
have included CTAB, Quiagen and low melt gel preps. Any other
suggestions or are these the same frequencies that everyone gets?
Thanks for all suggestions.