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)1.5231.976.176.0
Genomic DNA 7 (gRNA1d)1.5141.8875.7
Genomic DNA 7 (gRNA1d)1.5261.8976.3
Genomic DNA 8 (gRNA2d)0.3151.7915.715.6
Genomic DNA 8 (gRNA2d)0.3211.7416.1
Genomic DNA 8 (gRNA2d)0.3021.7515.1
Genomic DNA 9 (gRNA4d)2.0921.94104.6104.4
Genomic DNA 9 (gRNA4d)2.0831.94104.2
Genomic DNA 9 (gRNA4d)2.0871.93104.4
Genomic DNA 10 (-g)1.8891.9494.594.0
Genomic DNA 10 (-g)1.8821.9394.1
Genomic DNA 10 (-g)1.871.9493.5
Genomic DNA 10.7721.9338.639.0
Genomic DNA 10.7821.8939.1
Genomic DNA 10.7841.8339.2
Genomic DNA 22.171.96108.5108.2
Genomic DNA 22.1781.95108.9
Genomic DNA 22.1461.95107.3
Genomic DNA 31.0161.8950.850.6
Genomic DNA 31.021.951
Genomic DNA 31.0011.9450.1
Genomic DNA 41.3981.9169.968.7
Genomic DNA 41.351.9367.5
Genomic DNA 41.3761.968.8
Genomic DNA 54.1441.99207.2206.7
Genomic DNA 54.1792209
Genomic DNA 54.082204
Genomic DNA 64.9071.99245.4244.0
Genomic DNA 64.91.98245
Genomic DNA 64.8291.99241.5

qPCR: CRISPR on chromatin & mCherry


qPCR Master Reaction list

Reaction Templates Primers (Targets) Amplicon Method
1g20 +doxg020US-F/ g020US-R152CNV
2g20 +doxGAPDH B2CNV
3g20 -doxg020US-F/ g020US-R152CNV
4g20 -doxGAPDH B2CNV
5g29 +doxg029US-F/ g029US-R141CNV
6g29 +doxGAPDH B2CNV
7g29 -doxg029US-F/ g029US-R141CNV
8g29 -doxGAPDH B2CNV
9+doxg020US-F/ g020US-R152CNV
10+doxg029US-F/ g029US-R141CNV
11+doxGAPDH B2CNV
12-doxg020US-F/ g020US-R152CNV
13-doxg029US-F/ g029US-R141CNV
14-doxGAPDH B2CNV
151dq2F / q3R187CNV
161dGAPDH B2CNV
172dq1F / q1R142CNV
182dGAPDH B2CNV
194dq2F / P2R140CNV
204dGAPDH B2CNV
21mockq2F / q3R187CNV
22mockq1F / q1R142CNV
23mockq2F / P2R140CNV
24mockGAPDH B2CNV
  • 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




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