Haynes Lab:Notebook/CRISPR Editing/2014/08/03

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08/03/2014

  • Gal4EED/luc transfection
  • qPCR on luciferase - primer test
  • mCherry CRISPR - extract genomic DNA
  • mCherry CRISPR - flow cytometry



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 Mix tubes

  1. Gal4EED/luc genomic DNA (21 wells)
  2. nTc (dH2O) (21 wells)
Reagent (Single) x21.5
DNA (20 ng) or dH2O up to 4.5 ---
dH2O --- ---
Total 4.5 96.75


Primer Master Mix tubes

  1. gRNA20 ES (6 wells)
  2. gRNA20 US (6 wells)
  3. gRNA29 ES (6 wells)
  4. gRNA29 US (6 wells)
  5. GAPDH B2 (6 wells)
  6. MMP12 C4 (6 wells)
  7. GAPDH A3 (6 wells)
Reagent (Single) x6.5
2x Roche SYBR 7.5 48.75
750 nM F/R primers 3.0 19.5
Total 10.5 68.25


Results summary

Primers Ct for DNA nTc signal? Melt Peak Conclusion
gRNA20 ES ### no ### ###
gRNA20 US ### no ### ###
gRNA29 ES ### no ### ###
gRNA29 US ### no ### ###
GAPDH B2 ### no ### ###
MMP12 C4 ### YES ### ###
GAPDH A3 ### no ### ###



mCherry CRISPR - extract genomic DNA

  • Select samples form the Wednesday practical excercise
  • Used QIAmp genomic mini kit
  • Measured concentrations with Nanodrop
Sample OD260 260/280 ng/μL
1 0.499 1.95 24.9
2 0.924 1.95 46.2
4 1.020 1.99 51.0
Mock ("blank") 0.319 2.01 15.9



mCherry CRISPR - flow cytometry

  • Harvested cells from 80% confluent 6-well plates
  • Resuspended pellets in 1 mL cold FACS buffer (5% FBS, 1xPBS); strained through mesh caps
  • Guava HTS flow cytometer; load = 200 μL per well, 96-well round bottom well plate

Samples

  1. A1 - KAH154, no dox
  2. A2 - 1
  3. A3 - 2
  4. A4 - 4
  5. A5 - KAH154, +dox (overnight)




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