Used conjugation to introduce pBT20 (Himar-1 bearing GentR marker) to betaproteobacteria. Conducted a Southern of digested gDNA and got single bands (I think...it's kind of a crappy Southern, but I'm pretty sure) conducted arbitrary semirandom PCR to map insertions and all of the clones but one produced good sequence that mapped to appropriately sized fragments from the Southern. One clone would not produce decent sequence so I ligated the arbPCR fragments into pGEMT and transformed DH5a. I sequenced a few clones and each one looks like it may have a different insertion site. This doesn't make sense to me as some of the indicated fragments are bands that should be much different sizes than the one indicated in the Southern. Everything I've read about this system indicates that it is a stable single insertion system, but could there be a low frequency of these multiple insertion events?