NEB
NEBuilder® HiFi DNA Assembly Cloning Kit
E5520S
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My lab investigates the role of fatty acid transport proteins and the mechanism of fatty acid uptake into cells. As the mechanism is not well understood, we look for interaction partners for our protein. We needed to make a plasmid that expressed two fusion proteins from one promoter, each labelled with a different fluorescent protein and linked by an IRES sequence.
Plasmid construction with multiple fusion proteins
several plasmids
Be careful of primer design to get the correct junctions
The NEBuilder system made this relatively easy. PCR primers of 28 bases were used. The first 8 bases are complementary to the sequence of the end of the adjacent fragment, the other 20 bases match the end of the fragment to amplify. This leads to a 16 bp overlap at the ends of adjacent fragments and very efficient assembly of the desired construct in one step after the PCR fragments are produced. Nearly 1000% of tested plasmids were correct. Use of NEB Q5 polymerase leads to low levels of incorrect bases in the fragments and the included competent cells work very well, A version without cells is available.
Allows many fragments to be easily assembled as you want them to be
A bit expensive
Can assemble a really complex plasmid in one assembly step from PCR products and/or restriction fragments with the right primer design