Haynes Lab:Notebook/CRISPR Editing/2014/08/07

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Revision as of 08:41, 8 August 2014

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08/07/2014

  • CRISPR on chromatin: transfections of g21, g22, g27 into Gal4EED/luc cells
  • Luc-mCh swap: building the donor sequence
  • SURVEYOR assay - C-on-C and mCherry



CRISPR on chromatin: transfections

  1. gRNA21/Cas9, dox+
  2. gRNA22/Cas9, dox+
  3. gRNA27/Cas9, dox+
  4. gRNA21/Cas9, dox-
  5. gRNA22/Cas9, dox-
  6. gRNA27/Cas9, dox-
  • On 8/05/14, cells were grown in 4 mL P/S-free medium to ~80% confluence in a 6-well plate, 3 with 1 μg/mL dox, 3 without dox



Luc-mCh swap: building the donor sequence

  • Designed nested PCR primers to gradually build homology arms onto mCherry. The resulting donor should...
    • Place mCherry in-frame upstream of the luciferase gene
    • Add an ATG start codon onto mCherry (we had to use KAH154 as a template, where mCherry has no start)
    • Add a double stop (TAATAG) onto the end of mCherry
    • Add 130 bp of homology with TK-luciferase to each end



SURVEYOR assay
Assistance from Week 2 Team 2


Planning

Target Genomic DNA (template) Primers Amplicon (bp) Expected S-Cut fragments
mCherry gRNA1 CRISPR 1d qPCR 1F / P3R 329 ~229, 100
mCherry gRNA2 CRISPR 2d " 329 ~52, 277
mCherry gRNA4 CRISPR 4d " 329 ~180, 149
mCherry blank " 329 no cutting (PCR already done)
luciferase gRNA20 g20 +dox gRNA 021 US P F / gRNA 027 ES P R 1295 ~165, 1130
luciferase gRNA20 g20 -dox " 1295 ~165, 1130
luciferase gRNA29 g29 +dox " 1295 ~395, 900
luciferase gRNA29 g29 -dox " 1295 ~395, 900
luciferase mock +dox " 1295 no cutting
luciferase mock -dox " 1295 no cutting


PCR reaction

  • Reactions: 20 ng template DNA, 1x HF Phusion buffer, 0.2 mM dNTPs, 2.5 μL of 10 μM for each primer, 0.5 μL Phusion polymerase; in 50 μL final volume
  • Program
    • 98°C/ 30 sec
    • 30x [98°C/ 10 sec, 58°C/ 15 sec, 72°C/ 15 sec]
    • 72°C/ 10 min
    • 4°C hold


PCR product clean-up

  • Used QIAquick PCR Purification kit
  • Elute with 50 μL H2O
Sample OD 260 260/280 ng/uL
CRISPR 1d 1.562 1.81 78.1
CRISPR 2d 0.273 1.71 14.0
CRISPR 4d 1.77 1.588 79.3
g20 +dox 1.408 1.79 70.4
g20 -dox 1.057 1.78 52.9
g29 +dox 0.987 1.77 49.4
g29 -dox 0.856 1.82 42.8
+dox 1.300 1.77 65.0
-dox 1.169 1.8 58.5
Blank 1+3 --- --- ~22.0
  • Combined Blank 1 and Blank 3, ~22 ng/μL in 100 uL volume


Mismatch annealing

  • Testing 2 methods
    • IDT approach - mix the mutant PCR sample with a wild type reference sample to ensure heteroduplex formation
    • Zhang Lab approach - do not mix with a wild type reference, since ~50% or more of the amplicons in the mutated cell sample will be wild type anyway
  • 200 ng clean PCR, 1x HF Phusion buffer, in final volume of 15 μL
  1. 200 ng CRISPR "1" (Zhang approach)
  2. 200 ng Blank (wild type control)


  • Thermal cycler program
    • 95ºC/ 10 min
    • 95ºC -- (‐2.0 ºC/s) --> 85ºC
    • 85ºC/ 1 min
    • 85ºC -- (‐0.3 ºC/s) --> 75ºC
    • 75ºC/ 1 min
    • 75ºC -- (‐0.3 ºC/s) --> 65ºC
    • 65ºC/ 1 min
    • 65ºC -- (‐0.3 ºC/s) --> 55ºC
    • 55ºC/ 1 min
    • 55ºC -- (‐0.3 ºC/s) --> 45ºC
    • 45ºC/ 1 min
    • 45ºC -- (‐0.3 ºC/s) --> 35ºC
    • 35ºC/ 1 min  
    • 35ºC -- (‐0.3 ºC/s) --> 25 ºC
    • 25ºC/ 1 min
    • 4 ºC/ hold ∞ 




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