Pcr blunt topo vector

To generate UAS flies, we used Q5 hot-start high-fidelity DNA polymerase (New England Biolabs) to amplify the transcripts of each candidate gene from complementary DNA (cDNA) synthesized using the total RNA of w¹¹¹⁸ fly heads (extracted using MiniPrep kit; Zymo Research, R1054). For each candidate gene, we designed more than 2 pairs of PCR primers in the 5’ and 3’ untranslated regions and inserted all resulting PCR products into pCR-Blunt-TOPO vector (Thermo Fisher). The TOPO-transcript vectors of the same gene were sequenced and compared to verify that no error was introduced to the coding sequence during reverse transcription. The verified coding sequences were then amplified and assembled into either the pUAST-attB vector (Nep3) or a modified pUAST-attB vector in which a FLAG tag was added at the 3’ end (Dg, CG5027, DopR, Ostγ, and piezo). To generate Piezo mutants, myc tag was inserted into amino acid position 901, 2289, 2291, and 2306 (Figure 5; Figure S6) in plasmid pUAST-attB-piezo-FLAG using Q5 site-directed mutagenesis kit (New England Biolabs). Each plasmid was sequence confirmed by full-length DNA sequencing.
The pUAST-attB constructs were inserted into either attP40 or attP86Fb (for Piezo constructs) landing sites. G₀ flies were crossed to a white⁻ balancer, and all white⁺ progenies were individually balanced and verified by sequencing.