Haynes Lab:Notebook/CRISPR Editing/2015/02/13

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02/13/2015 Want to sequence to determine editing efficiencies for all samples. Will prep the ones I have PCRd already, g023 and g034: g023 and P173, distance 150 g034 and P176, distance 291 Sample name template Primer 1 Luc14 g023 1 P173 2 Luc14 g023 2 P173 3 Luc14 g023 3 P173 4 Gal4 g023 1 P173 5 Gal4 g023 2 P173 6 Gal4 g023 3 P173 7 Luc14 untreated P173 8 Luc14 untreated P173 Sample name template Primer 9 Luc14 g034 1 P176 10 Luc14 g034 2 P176 11 Luc14 g034 3 P176 12 Gal4 g034 1 P176 13 Gal4 g034 2 P176 14 Gal4 g034 3 P176 15 Luc14 untreated P176 16 Luc14 untreated P176 PCR of gDNA samples from gRNA treated samples: Cell type treatment rep ng/µL ul for 200ng 2.1rxns Luc14 g023 1 82.358 2.43 5.10 Luc14 g023 2 76.437 2.62 5.50 Luc14 g023 3 119.281 1.68 3.53 GAL4-EED +dox g023 1 122.844 1.63 3.42 GAL4-EED +dox g023 2 102.434 1.95 4.10 GAL4-EED +dox g023 3 101.862 1.96 4.12 Luc14 g045 1 181.634 1.10 2.31 Luc14 g045 2 246.109 0.81 1.71 Luc14 g045 3 174.603 1.15 2.41 GAL4-EED +dox g045 1 74.461 2.69 5.64 GAL4-EED +dox g045 2 82.614 2.42 5.08 GAL4-EED +dox g045 3 58.964 3.39 7.12 Luc14 g046 1 255.228 0.78 1.65 Luc14 g046 2 170.592 1.17 2.46 Luc14 g046 3 144.251 1.39 2.91 GAL4-EED +dox g046 1 72.999 2.74 5.75 GAL4-EED +dox g046 2 78.598 2.54 5.34 GAL4-EED +dox g046 3 77.323 2.59 5.43 Luc14 g048 1 172.85 1.16 2.43 Luc14 g048 2 217.50 0.92 1.93 Luc14 g048 3 190.13 1.05 2.21 GAL4-EED +dox g048 1 183.71 1.09 2.29 GAL4-EED +dox g048 2 189.83 1.05 2.21 GAL4-EED +dox g048 3 209.10 0.96 2.01 Luc14 Untreated 1 203.77 0.98 2.06 Luc14 Untreated 2 201.30 0.99 2.09 Luc14 Untreated 3 221.91 0.90 1.89 GAL4-EED +dox Untreated 1 205.61 0.97 2.04 GAL4-EED +dox Untreated 2 219.95 0.91 1.91 GAL4-EED +dox Untreated 3 205.91 0.97 2.04 Mastermix 1 ' 1rxn 50.6 H20 30.8 1558.48 HF 5X MasterMix 10 506 dNTPs 1 50.6 FP P197 2.5 126.5 RP P198 2.5 126.5 DMSO 1.5 75.9 Total 48.3 2443.98 Mastermix 2 ' 1rxn 2.1 rxns MM1 47.3 101.43 Template 2 4.2 Phusion 0.5 1.05 Total 50 Sending out for sequencing Sample name template Primer 17 Luc14 g023 1 P149 18 Luc14 g023 2 P149 19 Luc14 g023 3 P149 20 Gal4 g023 1 P149 21 Gal4 g023 2 P149 22 Gal4 g023 3 P149 23 Luc14 g045 1 P176 24 Luc14 g045 2 P176 25 Luc14 g045 3 P176 26 Gal4 g045 1 P176 27 Gal4 g045 2 P176 28 Gal4 g045 3 P176 29 Luc14 g046 1 P165 30 Luc14 g046 2 P165 31 Luc14 g046 3 P165 32 Gal4 g046 1 P165 33 Gal4 g046 2 P165 34 Gal4 g046 3 P165 35 Luc14 g048 1 P170 36 Luc14 g048 2 P170 37 Luc14 g048 3 P170 38 Gal4 g048 1 P170 39 Gal4 g048 2 P170 40 Gal4 g048 3 P170 41 Luc14 Unt 1 P149 42 Luc14 Unt 2 P149 43 Luc14 Unt 3 P149 44 Gal4 Unt 1 P149 45 Gal4 Unt 2 P149 46 Gal4 Unt 3 P149 47 Luc14 Unt 1 P176 48 Luc14 Unt 2 P176 49 Luc14 Unt 3 P176 50 Gal4 Unt 1 P176 51 Gal4 Unt 2 P176 52 Gal4 Unt 3 P176 53 Luc14 Unt 1 P165 54 Luc14 Unt 2 P165 55 Luc14 Unt 3 P165 56 Gal4 Unt 1 P165 57 Gal4 Unt 2 P165 58 Gal4 Unt 3 P165 59 Luc14 Unt 1 P170 60 Luc14 Unt 2 P170 61 Luc14 Unt 3 P170 62 Gal4 Unt 1 P170 63 Gal4 Unt 2 P170 64 Gal4 Unt 3 P170