At long last, I got all the flies together that we need for sufficient statistical power. As the preliminary data had indicated, WTB flies don’t learn with the short training, while rut and rsh flies do just fine.

However, this may be due to genetic background effects, so we need to check the CRISPR mutants.

DNA Assembly in a 1:2 ratio of vector to insert with gRNAs of rsh and rut (Q5 and template concentration: 640 pg/µl) and 100ng of pCFD6 BbsI AP (using QuickCIP). Heat-shock (hs) transformation into E. coli (DH5α competent) with 10 µl Assembly Reaction and 100 µl cells.

For Crtl, pCFD6 BbsI AP was wrongly used.

DNA Assembly in a 1:2 ratio of vector to insert with the gRNAs of rsh and rut (Q5 and template concentration: 640 pg/µl) and 100ng of pCFD6 BbsI AP (using FastAP and 2 extraction steps). Heat-shock (hs) transformation into E. coli (DH5α competent) with 10 µl Assembly Reaction and 100 µl cells.

For Crtl, pCFD6 BbsI AP was used in reaction.

Via gDNA analysis and PCR was the specific area of the rsh gene extracted and amplified where the nucleotide substitution: C to T (Folkers et al., 2006) should be for the rsh¹ mutation. The amplicon was Sanger sequenced which proved the nucleotide substitution.

Despite very warm weather, some flies did fly, even though the learning performance of the control flies was really poor. At least for now, it looks like all stocks are learning and that rut and rsh flies learn at least equally well as the Berlin flies. I’ve also managed to fix the positive preference problem: