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

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'''qPCR on luciferase - primer test'''
'''qPCR on luciferase - primer test'''


'''Templates Master Mixes'''
'''Templates Master Mix tubes'''
# Gal4EED/luc genomic DNA
# Gal4EED/luc genomic DNA
# nTc (dH<sub>2</sub>O)
# nTc (dH<sub>2</sub>O)
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{| {{table}}
{| {{table}}
|-
|-
| Reagent || (Single) || x6.5
| Reagent || (Single) || x21.5
|-
|-
| DNA (20 ng) or dH<sub>2</sub>O || up to 4.5 || ---
| DNA (20 ng) or dH<sub>2</sub>O || up to 4.5 || ---
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| dH<sub>2</sub>O || --- || ---
| dH<sub>2</sub>O || --- || ---
|-
|-
| Total || 4.5 || 29.5
| Total || 4.5 || 96.75
|}
|}






'''Primer Master Mixes'''
'''Primer Master Mix tubes'''
# gRNA20 ES
# gRNA20 ES
# gRNA20 US
# gRNA20 US
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{| {{table}}
{| {{table}}
|-
|-
| Reagent || (Single) || x21.5
| Reagent || (Single) || x6.5
|-
|-
| 2x Roche SYBR || 7.5 || 161.25
| 2x Roche SYBR || 7.5 || 48.75
|-
|-
| 750 nM F/R primers || 3.0 || 64.5
| 750 nM F/R primers || 3.0 || 19.5
|-
|-
| Total || 10.5 || 225.75
| Total || 10.5 || 68.25
|}
|}


Revision as of 10:52, 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 Mix tubes

  1. Gal4EED/luc genomic DNA
  2. nTc (dH2O)
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
  2. gRNA20 US
  3. gRNA29 ES
  4. gRNA29 US
  5. GAPDH B2
  6. MMP12 C4
  7. GAPDH A3
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




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