Haynes Lab:Notebook/CRISPR Editing/2014/08/05

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08/05/14

  • SURVEYOR assay - for mCherry CRISPR
  • qPCR melt curve optimization - mCherry CRISPR 1



SURVEYOR assay
Assistance from Week 2 Team 1

PCR primers


PCR templates

  1. CRISPR "1"
  2. CRISPR "2"
  3. CRISPR "4"
  4. "Blank" (3x)


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 ∞ 



qPCR melt curve optimization - mCherry CRISPR 1
Assistance from Kumaran Lleng

Optimization parameters

  • Borun et al reported cleaner peak profiles after a 30-cycle amplification (vs. 40 cycles)
    • We will use 30 cycles instead of 45
  • Borun et al used at LEAST 50 ng genomic DNA in their reactions (final amount per PCR ambiguous)
    • We will test 20 ng, 50 ng, and 70 ng (70 is our upper limit because of the concentration of the Blank DNA sample)


Master reaction list

Rxn # Template Target (primers) Method
11 20 ng q2F_q3RCNV melt
21 20 ngGAPDH B2"
31 50 ngq2F_q3R"
41 50 ngGAPDH B2"
51 70 ngP3F_q3R"
61 70 ngGAPDH B2"
7Blank 20 ngq2F_q3R"
8Blank 20 ngGAPDH B2"
9Blank 50 ngq2F_q3R"
10Blank 50 ngGAPDH B2"
11Blank 70 ngq2F_q3R"
12Blank 70 ngGAPDH B2"

Notes:

  • Opaque white plates
  • Final reaction volumes = 15 μL
  • Each reaction had 3.0 μL of 750 nM F/R primers, 20 ng - 70 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
  • nTc for q2F_q3R and GAPDH confirmed no background signal under identical conditions on 08/04/14


Run PCR - Bio-Rad CFX96

  • 95°C, 3 min
  • 30x [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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