on Monday, February 5th, 2018 11:01 | by Ottavia Palazzo
Maps of the FoxP gene and of the pTGem plasmid used for cloning: 1 desired construct is aiming to target all dFoxP isoforms with one homolgy domain starting at exon 1 to exon 3, the second homology domain starting at exon 3 to exon 6. The second construct is aimed to target just dFoxP IsoB, with both the homology domain at the end of the protein.
PCR product of the homology domains 1 and 2 for FoxP (upper part of the picture, left and right), and homology domains 1 and 2 for FoxP-isoformB (lower part of the picture, left and right): amplification of the desired homology domains via PCR (4 replicates for each construct in order to test different annealing temperatures). The products will be used for subsequent cloning in the vector.
Plasmid with Hom1 Hom2 FoxP ready: the image shows the presence of the homology domain 2 for dFoxP in a plasmid where we already ligated the homolgy domani 1. This construct is thus ready to be injected.
We transfected E.coli cells with the plasmids for IsoA and B from the Schaff lab (Berlin) and subsequently we sequenced them with 4 primers each (1 FW primer sequence in the plasmid upstream the ORF and 1 RV, 1 FW primer sequence in the middle of the ORF and 1 RV).
pcDNA plasmid IsoA correctly amplified in E.coli
pcDNA plasmid IsoB correctly amplified in E.coli