onetaq quick load 2x master mix  (New England Biolabs)

Journal: eLife

Article Title: Region-specific mechanosensation modulates Drosophila postural control behaviour

doi: 10.7554/eLife.108505

Figure Lengend Snippet: ( A ) Quantification of Hox RNA expression in larval sensory neurons. The flow diagram (beginning bottom left) shows how sensory neurons were collected from dissected first instar larvae using fluorescence-activated cell sorting (FACS). RNA was extracted from the sorted cells and reverse transcribed to DNA for amplification of Hox gene products via polymerase chain reaction (PCR). The photograph (bottom right) shows an agarose gel electrophoresis following RT-PCR of Hox genes Antp , Ubx , abd-A and Abd-B . For each gene, ‘no tem’ indicates a no template cDNA control, ‘no RT’ indicates a no reverse transcription control, and ‘exp’ indicates the experimental lane. ( B ) Self-righting times of first instar larvae expressing a Hox gene RNAi construct in the 109(2)80 domain. ***p < 0.001, *p < 0.05 for Wilcoxon rank sum tests, n = 19–30. ( C–F ) Confocal images of immunolabelled stage 16 109(2)80>mCD8::GFP embryos. In each case, the large left panel is a maximum intensity z-projection. The white square indicates the region that is zoomed in the smaller panels to the right. These smaller panels show an individual z slice in the two separate channels and the channel overlay. Triangles indicate cells showing clear signal for Hox protein, and arrowheads indicate cells lacking signal for Hox protein. The strips on the right are zoomed sections of the z-projection, showing a range of cells in one embryonic hemisegment. The putative identities of these cells are indicated by red lines in accordance with the canonical hemisegmental diagram on the far right. ( C ) Embryo immunolabelled for Antp and GFP, dorsal view. ( D ) Embryo immunolabelled for Antp and GFP, ventrolateral view. ( E ) Embryo immunolabelled for Abd-B and GFP, dorsal view. ( F ) Embryo labelled for Abd-B and GFP, ventrolateral view. Figure 6—source data 1. Original file of the full raw uncropped, unedited gel image depicting an agarose gel electrophoresis stained with ethidium bromide where the products of several RT-PCR reactions including no DNA template controls, no RT control and experimentals using primers specific for RP49 and the Hox genes Antennapedia , Ultrabithorax , abdominal A , and Abdominal B were resolved. Figure 6—source data 2. Labelled original file of the full raw uncropped, unedited gel image depicting an agarose gel electrophoresis stained with ethidium bromide where the products of several RT-PCR reactions including no template controls, no RT control and experimentals using primers specific for RP49 and the Hox genes Antennapedia , Ultrabithorax , abdominal A , and Abdominal B were resolved. Labels have been added indicating the relevant primer pairs used in each well (see bracket at the top) as well as the nature of the reaction (e.g. no DNA template, no RT control, experimental) for each primer pair. Amplicons (bands) of the expected size for each amplification reaction are labelled with asterisks.

Article Snippet: cDNA was then amplified via PCR using standard Taq DNA polymerase (NEB) for 40 cycles and with annealing temperature of 60°C.

Techniques: RNA Expression, Fluorescence, FACS, Reverse Transcription, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Construct, Confocal, Staining