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Generation of pENTR-promoter plasmids　Outline of promoter fusion gene construction

There are two major strategies to make a Gateway plasmid that carry the gene of interest (MyGene).

Strategy 1) The attR1-ccdB-attR2 cassette (Rf cassette) is inserted into your plasmid carrying MyGene by ligation. Rf cassette is commercially available from Invitrogen as "Gateway vector conversion system" (cat. 11828-019).
Note that your plasmid has to carry 3' regulatory sequences (such as a polyadenylation signal) that act in C. elegans to be compatible with this strategy.

Strategy 2) MyGene is cut out using restriction enzymes and ligated into the muticloning site (MCS) of pPD-DEST. pPD-DEST was generated by insertion of Rf cassette into pPD vectors by Andy Fire, and several versions have been prepaired (see C. elegans promoter/marker database)

(Important notice) Plasmids made in any of the above strategies carry the ccdB gene and therefore cannot propagate in standard E. coli strains. Use gyrA strains such as DB3.1 (Invitrogen, cat. 11782-018).