Haynes Lab:Notebook/CRISPR Editing/2015/07/28

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07/28/2015

  • mCherry qPCR/melt pre-course test (at CSH)



mCherry qPCR/melt pre-course test (at CSH)

  • The situation: In 2014, a single gBlock was created as the template to generate mutational donor sequences for CRISPR/HDR at different positions within mCherry in KAH154 cells. Double stops were added at positions 72, 162, and 255. The plan was to PCR-amplify three different donors to insert stops at each position in separate experiments. Because of a primer shipping delay, we could not generate these separate donors as planned, and instead used the whole gBlock as the donor. So, we now have an accidental experiment where the question is 'did all three stop-mutations get inserted in a single CRISPR treatment?'
  • mCherry donor gBlock map: https://benchling.com/s/1dsOuw/edit


Calculate number of wells

  • Templates (8 total):
    • From 2014: gDNA U-2 OS KAH154: CRISPR 1d, 2d, 4d
    • From 2015 (new): 3d (mixed population...transfected w/ p330-3T or pX330-3B + PCR donor Stop@162)
    • From 2015 (new): PCR'ed dsDNA donor Stop@72, Stop@162, Stop@255
    • No template control
  • Primer pairs (3 total):
    • P147/148 (flanks stop codons @72) = 148 bp; Tm ~60°C
    • P143/144 (...@162) = 100 bp; Tm ~55°C
    • P145/146 (...@255) = 100 bp; Tm = 57, 58.8°C
  • Number of unique reactions: 8 x 3 = 24 reactions
  • Number of wells needed: 24 rxns x 3 reps = 72 wells (1 plate)


Primer master mixes

  • Multiplier = 8 rxns x 3 reps + 3 = 27
  1. P147/148
  2. P143/144
  3. P145/146

Note: reference primers (GAPDH B2) are not here yet

Reagent Vol. μL (x27)
750 nM F/R primer mix 3.0 81.0
2x SYBR (Roche) 7.5 202.5
  10.5 283.5


Template master mixes (Nanodrop reads, ng/uL)

  • Note: Donor DNA are column-cleaned PCR products; make dilutions in case the concentrations are too high
  • Multiplier = 3 rxns x 3 reps + 3 = 12
  1. CRISPR 1d (135.9)
  2. CRISPR 2d (204.9)
  3. CRISPR 3d (11.3)
  4. CRISPR 4d (145.7)
  5. donor Stop@72 & 1:10 dilution (120.6 & 17.8)
  6. donor Stop@162 & 1:10 dilution (95.1 & 12.1)
  7. donor Stop@255 & 1:10 dilution (83.2 & 10.2)
  8. no template control (water)
Reagent Vol. μL (x12)
DNA (20 ng) 0.5 6.0
PCR H2O 4.0 48.0
  4.5 54.0

Loading

  • Template mix, 13.5 into each rep 1 well (A1, A4, A7...)
  • Primer mix, 31.5 into each rep 1 well (A1, B1, C1...H1...)

PCR - Bio-Rad CFX96

  • 95°C, 3 min
  • 40x [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec (measure)]
  • 72°C, 30 sec
  • Melt: 70°C/ 5 sec -- +0.1°C (measure) --> 95°C/ 5 sec


CONCLUSIONS

  • Overall, Ct values strangely very high for nTc and some templates (values of 10 - 24)
  •  ??? - P147/148 - no signal from Donor stop 1 control
  • P143/144 - appears to work well

TROUBLE SHOOTING

  • Use less DNA template



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