Haynes Lab:Notebook/CRISPR Editing/2014/08/03: Difference between revisions

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| Reagent | (Single) | x21.5
| Reagent | (Single) | x21.5
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| 2x Roche SYBR | 7.5 | 161.25
| 2x Roche SYBR | 7.5 | 161.25
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Revision as of 10:45, 4 August 2014

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08/03/2014

  • Gal4EED/luc transfection
  • qPCR on luciferase - primer test


Gal4EED/luc transfection

  • Lipofectamine LTX transfection of cells, 6-well plate format
  • Gal4EED/luc +dox from T-25 trypsinized and plated in wells 1-3 (4 mL medium)
  • Gal4EED/luc (no dox) from T-25 trypsinized and plated in wells 4-6 (4 mL medium)
    • Growth medium added back to original flask to grow up remaining cells as back-up
  • Transfection carried out as described in the CSH Syn Bio 2014 manual (2000 ng DNA/ 600 μL transfection mix/ 4 mL cell sample)
  1. Gal4EED/luc +dox, plasmid gRNA 20
  2. Gal4EED/luc +dox, plasmid gRNA 29
  3. Gal4EED/luc +dox, mock
  4. Gal4EED/luc (no dox), plasmid gRNA 20
  5. Gal4EED/luc (no dox), plasmid gRNA 29
  6. Gal4EED/luc (no dox), mock
  • Note: used complete DMEM (with pen/strep) so cell viability might be suboptimal because of this
  • 8/4/14 - carefully replaced original transfection medium with DMEM/10% FBS


qPCR on luciferase - primer test

'Templates Master Mixes

  1. Gal4EED/luc genomic DNA
  2. nTc (dH2O)
(Single) | x21.5
7.5 | 161.25
3.0 | 64.5
10.5 | 225.75


Primer Master Mixes

  1. gRNA20 ES
  2. gRNA20 US
  3. gRNA29 ES
  4. gRNA29 US
  5. GAPDH B2
  6. MMP12 C4
  7. GAPDH A3



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