Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/07/13

Transfection of Gal4EED-Luc HEK293

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Overview Going to perform four transfections of Gal4EED-Luc cell line HEK293 in 6-well plates, using four combinations of donor plasmid and gRNAs/Cas9 plasmids. g032 (gRNA #1), g044 (gRNA #2), DBN007 (donor) g034 (gRNA #1), g044 (gRNA #2), DBN007 (donor) g032 (gRNA #1), g048 (gRNA #2), DBN007 (donor) g034 (gRNA #1), g048 (gRNA #2), DBN007 (donor) Notes Plasmid concentrations: plasmid conc. (ng/µL) amt needed (ng) volume required (µL) g032 190 400 2.1 g034 180 400 2.2 g044 164 400 2.4 g048 240 400 1.7 DBN007 398 1200 3.0 Calculations for making plasmid solutions: Transfection gRNA 1 vol (µL) gRNA 2 vol (µL) donor vol (µL) water vol (µL) 1 2.1 2.4 3.0 12.5 2 2.2 2.4 3.0 12.4 3 2.1 1.7 3.0 13.2 4 2.2 1.7 3.0 13.1 I'll be performing transfections using the Lipofectamine LTX kit, following their instructions with one addition: centrifugation of the plate immediately post-transfection, 30 min @ 126 g and 37 °C. Plate centrifugation has been shown to greatly enhance transfection efficiency in other cell lines.