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

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08/06/2014

  • Genomic DNA extractions
  • qPCR: CRISPR on chromatin & mCherry (single plate)



Genomic DNA extractions

Samples

  1. HEK gRNA20/Cas9, +dox cells
  2. HEK gRNA20/Cas9, -dox cells
  3. HEK gRNA29/Cas9, +dox cells
  4. HEK gRNA29/Cas9, -dox cells
  5. HEK mock, +dox cells
  6. HEK mock, -dox cells
  7. U2OS 1d
  8. U2OS 2d
  9. U2OS 4d
  10. 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

  1. g020US-F/ g020US-R (12 wells)
  2. g029US-F/ g029US-R (12)
  3. q2F / q3R (6)
  4. q1F / q1R (6)
  5. q2F / P2R (6)
  6. GAPDH B2 (30)


Template Master Mixes

  1. g20+dox (6 wells)
  2. g20-dox (6)
  3. g29+dox (6)
  4. g29-dox (6)
  5. +dox (9)
  6. -dox (9)
  7. 1d (6)
  8. 2d (6)
  9. 4d (6)
  10. 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