XL1-Red in vivo mutagenesis

[neuro]

We are trying random mutagenesis of a 9kb plasmid using the XL1-Red in vivo 
mutagenesis system (Stratagene). Even if we follow the instructions of the 
manual, we can't find any mutations in the gene of interest. The gene, a 
restriction/modification system, is not essential for bacterial survival, 
except for a mutation effecting the modification function . The mutation 
frequency of the XL-1 Red  strain should be 1 per 2000 bp per 30 
generations, as ruled out by the suppliers. We sequenced more than 10kb 
without any mutation after 40-50 generations. The technical service of 
Stratagene has no explanation for this. Is there anybody having the same 
experience or some better. Or is there anybody having an idea for efficient 
random in vivo mutagenesis of a plasmid.

Andreas Meisel	
Exp. Neurology
Humboldt University Berlin
meisel at neuro.charite.hu-berlin.de