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

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| mCherry gRNA2 || CRISPR 2d || " || 329 || ~52, 277
| mCherry gRNA2 || CRISPR 2d || " || 329 || ~52, 277
|-
|-
-
| mCherry gRNA4 || CRISPR 2d || " || 329 || ~180, 149
+
| mCherry gRNA4 || CRISPR 4d || " || 329 || ~180, 149
|-
|-
| mCherry || blank || " || 329 || no cutting (PCR already done)
| mCherry || blank || " || 329 || no cutting (PCR already done)
|-
|-
-
| luciferase gRNA20 || g20 || gRNA 021 US P F / gRNA  027 ES P R || 1295 || ~165, 1130
+
| luciferase gRNA20 || g20 +dox || gRNA 021 US P F / gRNA  027 ES P R || 1295 || ~165, 1130
|-
|-
-
| luciferase gRNA29 || g29 || " || 1295 || ~395, 900
+
| luciferase gRNA20 || g20 -dox || " || 1295 || ~165, 1130
|-
|-
-
| luciferase || mock || " || 1295 || no cutting
+
| 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
|}
|}

Revision as of 13:01, 7 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 "1" --- 1.82 40.3
CRISPR "2" --- 1.62 4.7
CRISPR "4" --- 1.85 9.8
Blank 1 --- 1.80 22.9
Blank 2 --- 1.54 5.0
Blank 3 --- 1.85 21.5
  • 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. 100 ng CRISPR "1" + 100 ng Blank (IDT approach)
  2. 200 ng CRISPR "1" (Zhang approach)
  3. 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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