The stem-loop constructs for silencing mRNAs (~500nt) having no homology (>18nt) to other transcripts in T. brucei genome was used to generate the stem-loop constructs using primers listed above. Briefly, PCR product of part of the mRNA coding region was cloned into TOPO vector as per manufactures instruction and was used as a donor template for LR cloning with pTrypRNAiGate vector. The constructs expressing dsRNA were linearized with EcoRV and transfected into the parental cell line; the transformants were selected with 5μg/mL phleomycin. To check the silencing i tagged my protein with PTP (pC-PTP-PURO- This is the plasmid i used for tagging). I choose the same 500 nts which i choose for silencing and i amplified the region with corresponding restriction sites and ligated with my plasmid then i confirmed with sequencing. finally, i transfected). When I did the western analysis i can see full of non-specific bands. All the experiments procedures are absolutely fine. I suspect the designing could be a problem. Can you share your comments it would be helpful for me
