Hi all,
-- I often do simple cloning of unmodified PCR products using a suicide vector. I wonder if anyone has tried to express the T4 DNA ligase in vivo so that the
ligation could happen in vivo?
I have only seen this paper by Ren et al https://www.ncbi.nlm.nih.gov/pubmed/9305776
They express T4 ligase from a plasmid and they manage to recircularize linear plasmids efficiently
in a way that is dependent on T4 ligase expression.
They do some experiments with double digested vectors where they cut out an internal part of a marker.
They get this marker back in 9 out of 50 cases (if I understood correctly, Table 4 ) for a recABC background.
Since there are two orientations, this means that 18 out of 50 clones are recombinant.
With some kind of counter selection against empty clones (I use the pCAPs vector, works very well) and loading more insert, the frequency should be
so high that you do not need to look at perhaps 3-5 clones.
Did anyone try this or something similar? This would be great for DIY, since there would be no need to purify the ligase.
/bjorn
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