Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 239

Fig. 2 Generating a Pmir::gfp::unc-54 3 0 UTR reporter construct. (A) By conventional cloning. Use
primer A and B to amplify a 2 kb fragment upstream of mature miRNA or pre-miRNA hairpin and insert
the fragment into MCS of pPD95.75. (B) By PCR fusion. Use Primer A and B to amplify the promoter
region and primer C and D to amplify gfp::unc-54 3 0 UTR from pPD95.75. Primer B adds a 24-bp region
from MCS upstream of gfp gene. Anneal these two amplicons and use primer A* and D* to amplify the
fusion product. (C) By Multisite Gateway cloning. Amplify the promoter region and the reporter gene with
primers that introduce appropriate attB recombination sites and recombine PCR products with donor
plasmids pDONR-P4-P1R and pDONR201, respectively. Recombine the products pENTRY-Pmir and
pENTRY-gfp with pDEST-DD03. See text for details.