Trouble with fosmid clone (Tn10 contamination?)
rafamaldo at yahoo.com
Tue Mar 8 10:05:35 EST 2005
HI Kai Kamm,
I guess the sequence from the compnay includes some of the genome
secuence from your Cnidarian bug...
Tn10 must be present in some E. coli host, must not? You may be using
XL1 Blue or some derivative carrying a wild type Tn10, which can jump
from one place to another of the genome. If you have used the EPI300
strain from Epicentre, which is Tn10 free (at least they say that;
EPI300 has another transposon, Tn5 kanR, but is not related to Tn10),
Tn10 must come from manipulations from the company. Unless you have used
some Tn10 containing strain...
Now my hypothesis: the insertion of Tn10 in your fosmid happened after
you sent the clone to the company. Then, you retained a fresh, Tn10 free
fosmid and they got an insertion of Tn10 in the fosmid.
You may check it easily because it must be tetracycline sensitive, since
Tn10 confers resistance to tet. Other modified Tn10 have resistance to
cam or kan, but they are much very rare. Other option, expensive and
time consuming, is a southern blot using Tn10 as a probe. It would be
nice if you cauld get back the fosmid from the company and do the same.
In case you have been using some strain containing Tn10, it will be
TetR. Extract your fosmid and transform another strain sensitive to
tetracycline. Check now if the resistance is linked to your cosmid, wich
I guess is not since you checked that by restriction.
Transposition of Tn10 is rare, but no zero. That kind of problems arises
when working with live material... :)
Kai Kamm wrote:
> 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!
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