We have a couple of plasmids with large (about 20 kb)
inserts containing DNA subcloned from a maize YAC. Despite
having repeat regions in them, they have been rock stable in
plasmid form in the host strain DH5alpha. Further, I
extensively hopped tn1000 transposable elements into them by
the standard conjugation (in vivo) method. Again, I observed
no deletions among the approximately 200 of these I mapped.
Some time ago I sequenced most of these plasmid's insert
DNA using the tn1000's. But there were places in both
plasmids the tn1000's would not jump into for me.
Recently we have tried two in vitro transposition kits to
fill these two gaps. The "Primer Island" kit from PE (which
uses Ty1 integrase) and the Epicenter kit (which uses a
modified bacterial transposase.) Both seem to result in a
very high frequency of deletions. I'd estimate >80% have
deletions. The deletions make them pretty much impossible to
map. We are using DH10B electrocompetant cells to
electroporate into. Though I haven't used DH10B extensively
I have a hard time believing they are the source of the
deletions.
Has anybody had similar problems?
Phillip San Miguel
