Help on nested deletion system
drm21 at mole.bio.cam.ac.uk
Wed Oct 18 11:50:56 EST 1995
In article <460lpo$p9h at oac4.hsc.uth.tmc.edu>, sa95078 at odin.mda.uth.tmc.edu
>Hi, can anyone give me some suggestion for a reliable unidirectional
>nested deletion system.
>Thanks in advanced.
It depends what you want them for! I found the ExoIII-type procedure (eg
Erase-a-base) to be a complete pain in the ass. If you want to generate
the nested set for sequencing, then I REALLY (re-)recommend the
gamma-delta transposon (Tn1000) method described by Strathmann et al PNAS
88,1247-1250,1991. This method is unbelievably (IMO) quick and easy to
use, because bacteria do all the hard work.
Essentially, you allow the gamma-delta transposon (which is on the F
factor) to insert (randomly) into your sequence. In an appropriate (RecA+,
I'm not sure why) donor strain, the product of this insertion is a
co-integrate of the F-factor and your plasmid. Following mating of the F+
strain to an F- recipient strain, the co-integrate is resolved into an F
episome and your plasmid with a transposon inserted randomly into it.
So, you just transform your DNA into a special donor strain (F+, RecA+,
Amp(S), Kan(S) eg DPWC) and mate to a recipient strain (F-, RecA-, Amp(S),
Kan(R) eg BW26). The Kan(R)Amp(R) colonies which result from this mating
are your nested set - the site of transposon insertion can be mapped by
restriction enzyme analysis, or more easily by PCR direct from the
colonies. You can sequence off each end of the transposon, so that this
procedure allows complete double-strand sequencing.
I first heard of this method here on methods_reagents and can't believe
how well it worked. Much as I dislike kits in general, the only place I
was able to get hold of the necessary strains was from Gold Biotechnology
in their Tn1000 kit. (I have absolutely no affiliation with Gold
Biotechnology, and only bought the kit for the strains. It did work
extremely well, first time though!)
Wellcome/CRC Institute, Time flies like an arrow...
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