Transposon mutagenesis of Pseudomonas with Epicentre kit - Need a method for identifying the disrupted gene (Dec/02/2004 )

I have several mutants I created with the Epicentre EZ::TN transposon mutagenesis kit (kanamycin resistance cassette).

I am having problems sequencing the region around the inserted cassette ie identifying the gene that has been disrupted. There is very little detailed info available in the literature.

Epicentre suggests a direct sequencing method (directly from genomic DNA) but I tried this and it was not specific enough (sequence was all messed up).

I am currently trying a restriction digest, self ligation, PCR and sequencing method and am not getting anywhere fast. The primers for the PCR and sequencing come with the kit and amplify outwards from the cassette. I am using EcoR1 for the digest which does not cut the cassette.

Has anyone attempted this before and if so could you please share your method with me?

Thanks in advance

I am still stumped by this problem.

The way I understand it is:

Take bacterial genome containing transposon insert
Digest with RE (EcoR1)
Self-ligate using T4 DNA ligase overnight at 14 degrees.

This is the point I am up to now. When I run a gel of the digest and self-ligation I can't tell any difference (my supervisor says that is not unusual?). Is there another way to tell if the self-ligation worked?

I now have to amplify by PCR using primers supplied with the kit that amplify OUTWARDS from the insert.. At the same time I need to optimise the PCR but effectively have no positive control available (hmm fun).

First PCR = tried 4 mutants, at all 3 temps tested (50,55,60) there was a small faint band right at the top of the lane (but below the well) which I am guessing is either undigested DNA (is that possible in a PCR reaction? I would have loaded approximately 10ng DNA in the gel if the PCR had failed) or a very large product... Also there were 3 products of size approx 150bp, 300bp and 400bp in 3 of the mutants (2 at 60 degrees, the other one at 55 degrees) - would this be what I am looking for?

One idea I had was that maybe the amount of DNA in my PCR was inhibitory - is 10ng likely to be too much DNA?? Should I dilute it 1 in 10?

Also can you re-PCR the left over product from the first PCR if it contains loading buffer? So if I find the product I want and need more of it for sequencing, do I need to re-do it because the loading buffer can't go into the thermocycler?

I am not a strong mol biol student therefore I really have no idea what to do right now!!

Anyone have a suggestion???