Trouble with fosmid clone (Tn10 contamination?)

Kai Kamm kai.kamm at ecolevol.de
Sun Mar 6 12:04:19 EST 2005


Hi there

I have a little trouble with one of my fosmid clones (copy control 
epicentre). To save some time and nerves I sent one of my clones to a 
sequencing company.  What they sent me back was four contigs, two of them 
containing most of the 13,5kb E.coli Transposase Tn10 (except 500bp in a 
subcloning gap). Well, I compared the sequences with my restriction digests, 
did some new digests (with old and new preps) and realized that there are no 
restriction fragments of the Tn10 on my gels. In other words, the 
restriction pattern is only consistent with the sequence when  I remove the 
Tn10 from the contigs. Additionally, my digests (hindIII and HindIII/EcoRI) 
suggest a size of 40kb to 42kb maximum of the fosmid. The contigs of the 
company are already 48kb with still some subcloning gaps. There is still a 
big gap within a restriction fragment so that this clone would be at least 
50kb. The company didn't admit that it was their fault, in fact we didn't 
discuss who's fault it could have been, but they offered to do a new 
subcloning library. My digests seem unambiguous, so that my clone does not 
carry the Tn10 but I still fear that there could be something wrong with my 
clone. I was thinking of doing a PCR with a forward primer lying in the 
sequence of my species and a reverse primer lying in the Tn10 sequence. On 
the other hand, what would this PCR tell me? If there was no product OK, if 
there was a product... just more confusion because the restriction fragments 
of Tn10 are not there. Any suggestions what I should do or try? Anyone who 
has some knowledge about bacterial transposases and contamination of genomic 
libraries? I don't want to waste some thousend euros for the sequencing. On 
the other hand I want to have this clone done. The genomic library of my 
species (Cnidarian, 70% AT) and the subcloning of the clones cost me enough 
time and nerves.

Thanks a lot! 





More information about the Methods mailing list