No background plasmid
Roderic at biogene.co.uk
Sat Nov 1 05:46:19 EST 1997
In article <01bce651$70434ed0$1c524f92 at p823>, CHMC <adam at chmc.org>
>I've been looking for a plasmid that has a multiple cloning site in the
>middle of a suicide gene, so that an insert is nessesary for the
>transformants to survive. So far the only one that I've seen is one made
>by Invitrogen, which I am told you can't plasmid prep yourself, but have to
>by it from Invitrogen every time you want to use it.
I have used 2 positive selection plasmid vectors pCR-TRAP and pUN121.
Both rely on inserts conferring tetracycline resistance on transformants
as a result of derepressing Tet expression by disruption of a represor
encoding gene cI.
pCR-TRAP is a commercially available vector which comes as part of a kit
for cloning the PCR products. The kit is produced by GenHunter
Corporation (http://www.nashville.net/~genhunt) as part of their
differential display product range, we distribute their products in the
pUN121 is a vector in the public domain which I have used for library
construction in the past, it contains the following restriction sites in
the cI gene: BclI, SmaI, HindIII, EcoRI. The vector was developed by B.
Nilsson et al and is described in the following publication:
Nilsson B, Uhlen M, Josephson S, Gatenbeck S, Philipson L. (1983)
An improved positive selection plasmid vector constructed by
oligonucleotide mediated mutagenisis.
Nucleic Acids Research 11:8019-8030
The fact that tetracycline acts to supress growth of sensitive bacteria
as opposed to killing them means that the choice of bacterial strain and
tetracycline concentration in growth media is important. The commercial
pCR-TRAP kit comes with full instructins and a suitable host. pUN121
gave good results in DH10B, and 5K, although I never tried HB101 which
was the strain used by Nilsson et al. I found that the optimum
tetracycline concentration was 10ug/ml for 5K, which yeilded 93%
recombinants and a cloning efficiency of 1.5E4 recombinants per nanogram
of input fragment ( size selected genomic DNA fragments ranging from 2.4
to 2.5 kb in length ).
Roderic Fuerst E-mail: Roderic at biogene.co.uk
Bio/Gene Ltd Voice: +44 (0) 1480 861 831
6 The Business Centre Fax: +44 (0) 1480 861 899
Cambs. PE18 0NJ
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