TN5 library - (Nov/14/2009 )

I'm going to make a insertion library in E. coli using TN5 transposon ...does any one of you know if there will be multiple insertions using TN5? I'm not sure ...but as TN5 was used for sequencing long times ago i would suggest that there will be only one insertion per clone, but i'm not sure ...maybe someone of you knows more about that issue?

Many thanks in advance!

Regards,
p

Typically there is only a single insertion site, although rarely (< 1%) I have seen multiple. You can easily tell if you are sequencing for location of the site, since you get multiple overlapping reads during the sequencing, or multiple bands on a southern when probing.

Thanks a lot for your answer!

Does the 1% refer to the TN5 transposon or is this a general number?

Do you have an idea how multiple insertions are prevented?

Many thanks in advance!

Regards,
p

I have no experience with other transposons. I should mention that my work was with transposome electroporation (Epicentre, Goryshin, Reznikoff) rather than with plasmid carried copies of Tn5. I think the general efficiency of the transposome transformation is sufficiently low that duplicate insertions are rare. This may not be true if you have a multi-copy plasmid carrying the transposon. The transposome did not carry a copy of the transposase, so once an insertion happened, it was stable.

Thanks! I'm planning to use the TN5 Transposome as well!
You helped me a lot! Thanks for that!

Regards,
p