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1. Digestion of total cellular DNA with one or more restriction enzymes and
ligation of restriction half-site specific adaptors to all restriction fragments.
Select the bacterial genera you want to work with. A form will appear where we
may select the species, and the restriction enzymes and selective nucleotides for
the experiment.
We may also include the plasmids if available. For bacterial species with two
chromosomes, both chromosomes will be used in the experiment. In some
bacterial species, chromosomes are linear, and this fact has also been considered
in the experiment.
5'-
AG 3000 GT
-3'
Select restriction enzymes from the lists (restriction enzyme 1 from RE1 list,
and restriction enzyme 2 from RE2 list). They will be used to perform a
complete theoretical DNA digestion of the genome. Selective nucleotides may
also be introduced (see selective nucleotides in the form above: "AG" in the 5'
end, and "GT" in the 3' end).
The restriction enzymes available in the form have the following characterictics:
Selective nucleotides introduced in the form must match the upper strand of
DNA (see the grey segments in the picture).
The sequence of these adaptors are partially defined in the response page. Some
nucleotides of the adaptors are defined by the recognition sequence of the
restriction enzymes (in pale blue and pale green). The sequence of adaptors
which does not match the endonuclease recognition sequence (in magenta and
red) must be designed to avoid recognition of genomic DNA of the species used
in the experiment and to prevent aberrant results.
If the restriction enzymes used to cut the genomic DNA are not heat labile, or
restriction and ligation are performed simultaneously, the adaptor sequence must
not regenerate the original recognition sequence. To avoid this regeneration they
must be used adaptors mustbe used that produce a base change in the recognition
sequence. An example is shown below:
AFLP-PCR with primers matching adaptors, restriction enzymes recognised sequences and
containning aditional selective bases in the 3' end. Usage of selective primers will reduce
the number of bands amplified.