Haynes Lab:Notebook/CRISPR Editing/2014/08/06

Project name

Main project page Previous entry      Next entry

08/06/2014 Genomic DNA extractions qPCR: CRISPR on chromatin & mCherry (single plate) Genomic DNA extractions Samples HEK gRNA20/Cas9, +dox cells HEK gRNA20/Cas9, -dox cells HEK gRNA29/Cas9, +dox cells HEK gRNA29/Cas9, -dox cells HEK mock, +dox cells HEK mock, -dox cells U2OS 1d U2OS 2d U2OS 4d U2OS mock HEK = Gal4EED/luc cells U2OS = KAH154 CRISPR cells Sample OD 260 260/280 ng/uL Average ng/uL Genomic DNA 7 (gRNA1d) 1.523 1.9 76.1 76.0 Genomic DNA 7 (gRNA1d) 1.514 1.88 75.7 Genomic DNA 7 (gRNA1d) 1.526 1.89 76.3 Genomic DNA 8 (gRNA2d) 0.315 1.79 15.7 15.6 Genomic DNA 8 (gRNA2d) 0.321 1.74 16.1 Genomic DNA 8 (gRNA2d) 0.302 1.75 15.1 Genomic DNA 9 (gRNA4d) 2.092 1.94 104.6 104.4 Genomic DNA 9 (gRNA4d) 2.083 1.94 104.2 Genomic DNA 9 (gRNA4d) 2.087 1.93 104.4 Genomic DNA 10 (-g) 1.889 1.94 94.5 94.0 Genomic DNA 10 (-g) 1.882 1.93 94.1 Genomic DNA 10 (-g) 1.87 1.94 93.5 Genomic DNA 1 0.772 1.93 38.6 39.0 Genomic DNA 1 0.782 1.89 39.1 Genomic DNA 1 0.784 1.83 39.2 Genomic DNA 2 2.17 1.96 108.5 108.2 Genomic DNA 2 2.178 1.95 108.9 Genomic DNA 2 2.146 1.95 107.3 Genomic DNA 3 1.016 1.89 50.8 50.6 Genomic DNA 3 1.02 1.9 51 Genomic DNA 3 1.001 1.94 50.1 Genomic DNA 4 1.398 1.91 69.9 68.7 Genomic DNA 4 1.35 1.93 67.5 Genomic DNA 4 1.376 1.9 68.8 Genomic DNA 5 4.144 1.99 207.2 206.7 Genomic DNA 5 4.179 2 209 Genomic DNA 5 4.08 2 204 Genomic DNA 6 4.907 1.99 245.4 244.0 Genomic DNA 6 4.9 1.98 245 Genomic DNA 6 4.829 1.99 241.5 qPCR: CRISPR on chromatin & mCherry qPCR Master Reaction list Reaction Templates Primers (Targets) Amplicon Method 1 g20 +dox g020US-F/ g020US-R 152 CNV 2 g20 +dox GAPDH B2 CNV 3 g20 -dox g020US-F/ g020US-R 152 CNV 4 g20 -dox GAPDH B2 CNV 5 g29 +dox g029US-F/ g029US-R 141 CNV 6 g29 +dox GAPDH B2 CNV 7 g29 -dox g029US-F/ g029US-R 141 CNV 8 g29 -dox GAPDH B2 CNV 9 +dox g020US-F/ g020US-R 152 CNV 10 +dox g029US-F/ g029US-R 141 CNV 11 +dox GAPDH B2 CNV 12 -dox g020US-F/ g020US-R 152 CNV 13 -dox g029US-F/ g029US-R 141 CNV 14 -dox GAPDH B2 CNV 15 1d q2F / q3R 187 CNV 16 1d GAPDH B2 CNV 17 2d q1F / q1R 142 CNV 18 2d GAPDH B2 CNV 19 4d q2F / P2R 140 CNV 20 4d GAPDH B2 CNV 21 mock q2F / q3R 187 CNV 22 mock q1F / q1R 142 CNV 23 mock q2F / P2R 140 CNV 24 mock GAPDH B2 CNV 72 wells Primer Master Mixes g020US-F/ g020US-R (12 wells) g029US-F/ g029US-R (12) q2F / q3R (6) q1F / q1R (6) q2F / P2R (6) GAPDH B2 (30) Template Master Mixes g20+dox (6 wells) g20-dox (6) g29+dox (6) g29-dox (6) +dox (9) -dox (9) 1d (6) 2d (6) 4d (6) mock (12) Notes: Opaque white plates Final reaction volumes = 14 μL Each reaction had 3.0 μL of 750 nM F/R primers, 50 ng template DNA, 1x Roche SYBR Batch (master) mixes... Primers: Primers plus SYBR, 10.5 μL mix per relevant well, one mix per unique primer pair Templates: DNA plus water, 4.5 μL mix per relevant well, one mix per unique template Final triplicates for the 96-well plate were first made as a single batch, then aliquoted from the batch tube into the 96-well plate previous nTc for g020US-F/ g020US-R, g029US-F/ g029US-R, q2F / q3R, q1F / q1R, q2F / P2R, and GAPDH B2 confirmed no background signal under identical conditions Run PCR - Bio-Rad CFX96 95°C, 3 min 40x [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec (measure)] 72°C, 30 sec Melt: 70°C -- +0.1°C/ 5 sec (measure) --> 95°C