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

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(08/07/2014)
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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
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* SURVEYOR for C-on-C and mCherry
  
  
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** Add a double stop (TAATAG) onto the end of mCherry
 
** Add a double stop (TAATAG) onto the end of mCherry
 
** Add 130 bp of homology with TK-luciferase to each end
 
** Add 130 bp of homology with TK-luciferase to each end
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 +
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----
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'''SURVEYOR assay for C-on-C and mCherry'''
  
  

Revision as of 08:01, 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 for 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 for C-on-C and mCherry