Haynes Lab:Notebook/CRISPR Editing/2014/08/07: Difference between revisions

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




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'''SURVEYOR assay for C-on-C and mCherry'''
'''SURVEYOR assay'''<br>
Assistance from Week 2 Team 2
 
PCR primers
 
{|
|-
| Target || Genomic DNA (template) || Primers || Amplicon
| mCherry gRNA1d || CRISPR 1d || q1F / P3R || 329 bp
|}
 
* Annotated map at https://benchling.com/cshlsynbio/mammalian-crispr/Rc6-kah154/edit
 
 
'''PCR templates'''
# CRISPR "1"
# CRISPR "2"
# CRISPR "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 H<sub>2</sub>O
 
{| {{table}}
|-
| 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
 
# 100 ng CRISPR "1" + 100 ng Blank (IDT approach)
# 200 ng CRISPR "1" (Zhang approach)
# 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 ∞ 
 
 




Revision as of 09:09, 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

PCR primers

Target Genomic DNA (template) Primers Amplicon mCherry gRNA1d CRISPR 1d q1F / P3R 329 bp


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 ∞ 




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