Biomedical Engineering Reference
In-Depth Information
Fig.
1
General Multisite Gateway cloning scheme. This diagram presents a
schematic representation of a theoretical 3-fragment Multisite Gateway reaction
to link a promoter, N-terminal fusion tag, and gene of interest. The diagram iden-
tifi es the different plasmid types found in the BP and LR reactions as discussed
in the text
various confi gurations, and Fig.
1
shows a diagram of one of the
most common cloning schemes used for protein Expression clones.
You can use alternative confi gurations of the system if necessary as
long as you ensure that the leftmost and rightmost
attL
sites from
the Entry clones match the
attR
sites in the Destination vector,
and that each Entry clone junction contains an identical
attL
and
attR
specifi city to allow proper recombination.
DNA sequences of interest can be inserted into Entry clones in
several different ways, but to provide the most fl exibility in the
cloning strategy, we generally use a Gateway recombination reac-
tion (called the BP reaction,
see
Subheading
3.3
) to construct
Entry clones. Once Entry clones are constructed and sequence-
verifi ed, a second recombination reaction (called an LR reaction,
see
Subheading
3.4
) is used to carry out the subcloning into the
fi nal Destination vector. Destination vectors contain
attR
sites
fl anking a Gateway “cassette” which encodes a chloramphenicol
resistance gene and a toxin called
ccdB
. This region is recombined
out during the LR reaction and is replaced by the fragments from
the various Entry clones. A combination of positive selection using
the marker on the Destination vector and negative selection from
