Haynes Lab:Notebook/CRISPR Editing/2015/02/02

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02/02/2015 PCR of gRNA from Luc14 and GAL4-EED+dox samples, g034 Cell type treatment rep Concentration ul for 200ng ul for 800ng Luc14 g034 1 71.774 2.79 11.15 Luc14 g034 2 117.513 1.70 6.81 Luc14 g034 3 108.009 1.85 7.41 GAL4-EED +dox g034 1 86.013 2.33 9.30 GAL4-EED +dox g034 2 105.127 1.90 7.61 GAL4-EED +dox g034 3 122.48 1.63 6.53 Mastermix 1 ' 1rxn 25.5 H20 30.8 785.4 HF 5X MasterMix 10 255 dNTPs 0.2 5.1 FP 2.5 63.75 RP 2.5 63.75 Total 46 1173 Mastermix 2 ' 1rxn 4.2rxns MM1 46 193.2 Template 2 8.4 DMSO 1.5 6.3 Phusion 0.5 2.1 Total 50 205 Transfer 50μL of MM2 into each of 4-PCR tubes. PCR setup PCR 98°C for 3 min 30 cycles 98°C for 10s 69.5°C for 30s 72°C for 60s 72°C for 10 min 4°C forever Purified with Sigma genelute PCR clean up kit. Yields aren't as high as I'd like but I can work with it, especially if it reduces my background. Cell type treatment rep 260 280 260/280 ng/µL Luc14 g034 1 0.029 0.017 1.75 29.049 Luc14 g034 2 0.029 0.016 1.839 28.848 Luc14 g034 3 0.039 0.021 1.835 39.051 GAL4-EED +dox g034 1 0.039 0.021 1.841 38.915 GAL4-EED +dox g034 2 0.038 0.019 2.028 37.778 GAL4-EED +dox g034 3 0.042 0.022 1.914 42.041