For PCR verification of RMCE integration events, DNA was extracted from 10 to 15 adult flies using the PureLink™ Genomic DNA Mini Kit (Invitrogen). PCR was performed with tag-specific primers and MiMIC specific primers. Tag-specific primers (Tag-F and Tag-R) are mCherry-Seq-F and mCherry-Seq-R for mCherry, EGFPdo-Seq-F and EGFPdo-Seq-R for EGFP, EBFP2do-Seq-F and EBFP2do-Seq-R for EBFP2, TagRFPdo-Seq-F and TagRFPdo-Seq-R for TagRFP, Hrpdo-Seq-F and Hrpdo-Seq-R for HRP, Dendrado-Seq-F and Dendrado-Seq-R for Dendra, Killerreddo-Seq-F and Killerreddo-Seq-R for Killerred, GAL4-1R and GAL4-5F for GAL4, FLP0-Seq-R and SV40pA-Long-F for Flpo, and QF-Seq-R1 and Hsp70-pA-Alt-F for QF. MiMIC specific primers are Orientation-MiL-F and Orientation-MiL-R. PCR reaction conditions were: 1 μl DNA, 1 μl primer 1, 1 μl primer 2, 2 μl 10× Buffer, 0.16 μl dNTPs (25 mM each), 0.08 μl Qiagen HotStarTaq DNA Polymerase (QIAGEN), 14.76 μl milliQ water. PCR cycling conditions in PTC-225 or DNA Engine (MJ Research) were: denaturation at 94° for 10 minutes, 40 cycles at 94° for 30 seconds, 60° for 30 seconds and 72° for 60 seconds, and post-amplification extension at 72° for 10 minutes.
For each RMCE event, 4 PCR reactions were performed: a first PCR reaction with primers Orientation-MiL-F and Tag-R, a second PCR reaction with primers Orientation-MiL-F and Tag-F, a third PCR reaction with primers Orientation-MiL-R and Tag-R, and a fourth PCR reaction with primers Orientation-MiL-R and Tag-F. Since the transposon integrates one or two orientations relative to the gene, only one in two RMCE events is productive with respect to creating a gene trap or protein trap, which is reflected in a positive PCR for reactions 1 and 4, or 2 and 3. A “1/4” PCR pattern is always desired for a productive RMCE event (for example a gene or protein trap), when the gene/transposon configuration is 1/1 or −1/−1. Conversely, a “2/3” PCR pattern is diagnostic of a productive RMCE event, when the gene/transposon configuration is 1/−1 or −1/1. The reverse holds for unproductive RMCE events (Supplementary Figure 2 ).
For each RMCE event, 4 PCR reactions were performed: a first PCR reaction with primers Orientation-MiL-F and Tag-R, a second PCR reaction with primers Orientation-MiL-F and Tag-F, a third PCR reaction with primers Orientation-MiL-R and Tag-R, and a fourth PCR reaction with primers Orientation-MiL-R and Tag-F. Since the transposon integrates one or two orientations relative to the gene, only one in two RMCE events is productive with respect to creating a gene trap or protein trap, which is reflected in a positive PCR for reactions 1 and 4, or 2 and 3. A “1/4” PCR pattern is always desired for a productive RMCE event (for example a gene or protein trap), when the gene/transposon configuration is 1/1 or −1/−1. Conversely, a “2/3” PCR pattern is diagnostic of a productive RMCE event, when the gene/transposon configuration is 1/−1 or −1/1. The reverse holds for unproductive RMCE events (
