Haynes Lab:Notebook/CRISPR Editing/2014/08/04

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08/04/14

  • mCherry CRISPR qPCR
  • Gal4EED/luc CRISPR - change growth medium to pen-strep-free, with 1 μg/mL dox for dox+ cells



mCherry CRISPR qPCR
Genomic DNA from CRISPR transfected-cells:

  1. "1" - gRNA1/Cas9
  2. "2" - gRNA2/Cas9
  3. "4" - gRNA4T/Cas9n, gRNA4B/Cas9n
  4. "Blank" - mock transfection with H2O instead of plasmid

Master reaction list

Rxn # Template Target (primers) Method
1 1 q2F_q3R CNV melt
2 1 P3F_q3R CNV melt
3 1 GAPDH B2 CNV melt
4 Blank q2F_q3R CNV melt
5 Blank P3F_q3R CNV melt
6 Blank GAPDH B2 CNV melt
7 nTc q2F_q3R CNV melt
8 nTc P3F_q3R CNV melt
9 nTc GAPDH B2 CNV melt
10 4 q2R_q2F US specific
11 4 P3F_q3R US specific
12 4 GAPDH B2 US specific
13 Blank q2R_q2F US specific
14 Blank P3F_q3R US specific
15 Blank GAPDH B2 US specific
16 nTc q2R_q2F US specific
17 nTc P3F_q3R US specific
18 nTc GAPDH B2 US specific
19 2 q1F_q1R CNV melt
20 2 GAPDH B2 CNV melt
21 4 q2F_P2R CNV melt
22 4 GAPDH B2 CNV melt
23 Blank q1F_q1R CNV melt
24 Blank q2F_P2R CNV melt
25 Blank GAPDH B2 CNV melt
26 nTc q1F_q1R CNV melt
27 nTc q2F_P2R CNV melt
28 nTc GAPDH B2 CNV melt

Notes:

  • Opaque white plates
  • Final reaction volumes = 15 μL
  • Each reaction had 3.0 μL of 750 nM F/R primers, 20 ng template DNA, 1x Roche SYBR
  • Batch (master) mixes...
    • Primers: Primers plus SYBR, 10.5 μL mix per relevant well, one mix per unique primer pair
    • Templates: DNA plus water, 4.5 μL mix per relevant well, one mix per unique template (water-only for nTc)
    • Final triplicates for the 96-well plate were first made as a single batch, then aliquoted from the batch tube into the 96-well plate


Run PCR - Bio-Rad CFX96

  • 95°C, 3 min
  • 45 x [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec]
  • 72°C, 30 sec
  • Melt: 60°C -- +0.2°C/ 5 sec --> 95°C


Results

  • Hypothesis 1 - CNV melt: CRISPR editing will result in a lower loading-normalized peak height value, compared to untreated cells
  • Hypothesis 2 - US-specific amplification: CRISPR editing will result in a lower loading-normalized Cq value, compared to untreated cells

CRISPR 1 - CNV melt

  • Primer pair 1 (q2F_q3R), Temp = 86.60
    • Untreated control Peak_q2F_q3R / Peak_GAPDH = 5273.00 / 2959.00 = 1.78
    • CRISPR treated Peak_q2F_q3R / Peak_GAPDH = 5124.00 / 2902.00 = 1.77
    • Conclusion: Hypothesis is supported, with reservation (small value, no replicates)
    • Notes: no signal from nTc, so good primer pair with no primer dimer signal

CRISPR 1 - CNV melt

  • Primer pair 2 (P3F_q3R)
    • Untreated control Peak_P3F_q3R / Peak_GAPDH = 4922.00 / 2959.00 = 1.66
    • CRISPR treated Peak_P3F_q3R / Peak_GAPDH = 4693.00 / 2902.00 = 1.62
    • Conclusion: Hypothesis is supported, with reservation (small value, no replicates)
    • Notes: some signal from nTc, not-so-good primer pair

CRISPR 2 - CNV melt

  • Primer pair (q1F_q1R), Temp = 87.53
    • Untreated control Peak_q1F_q1R / Peak_GAPDH = 2361.38 / 2481.73 = 0.952
    • CRISPR treated Peak_q1F_q1R / Peak_GAPDH = 2292.12 / 2401.67 = 0.954
    • Conclusion: Hypothesis 1 rejected
    • Notes: no signal from nTc, so good primer pair with no primer dimer signal

CRISPR 4 - CNV melt

  • Primer pair (q2F_P2R), Temp = 87.20
    • Untreated control Peak_q2F_P2R / Peak_GAPDH = 2270.05 / 2481.73 = 0.92
    • CRISPR treated Peak_q2F_P2R / Peak_GAPDH = 2970.81 / 2548.25 = 1.16
    • Conclusion: Hypothesis 1 rejected
    • Notes: no signal from nTc, so good primer pair with no primer dimer signal

CRISPR 4 - US-specific

  • Primer pair 1 (q2R_q2F)
    • Untreated control 2^(Cq_q2R_q2F - Cq_GAPDH) = 2^(39.38 - 36.43) = 7.73
    • CRISPR treated 2^(Cq_q2R_q2F - Cq_GAPDH) = 2^(39.06 - 35.51) = 11.71
    • Normalized CRISPR treated value = 11.71 / 7.73 = 1.52
    • Conclusion: Hypothesis 2 is rejected
    • Notes: no signal from nTc, so good primer pair with no primer dimer signal

CRISPR 4 - US-specific

  • Primer pair 2 (P3F_q3R)
    • Untreated control 2^(Cq_GAPDH - Cq_P3F_q3R) = 2^(36.43 - 27.37) = 533.74
    • CRISPR treated 2^(Cq_GAPDH - Cq_P3F_q3R) = 2^(35.51 - 27.36) = 284.05
    • Normalized CRISPR treated value = 0.53
    • Conclusion: Hypothesis 2 is supported, with reservation
    • Notes: some signal from nTc (Cq ~42), not-so-good primer pair


Summary Table

Experiment Method Norm Expt. value Norm control Conclusion
"1" - gRNA1/Cas9, primer pair 1 CNV melt 1.77 1.78 Hypothesis 1 supported*
"1" - gRNA1/Cas9, primer pair 2 CNV melt 1.62 1.66 Hypothesis 1 supported*
"2" - gRNA2/Cas9 CNV melt 0.96 0.92 Hypothesis 1 rejected
"4" - gRNA4T/Cas9n, gRNA4B/Cas9n CNV melt 1.16 0.92 Hypothesis 1 rejected
"4" - gRNA4T/Cas9n, gRNA4B/Cas9n primer pair 1 US-specific 11.71 7.73 Hypothesis 2 rejected
"4" - gRNA4T/Cas9n, gRNA4B/Cas9n preimer pair 2 US-specific 284.05 533.74 Hypothesis 2 supported*

Note: *Hypothesis supported, with reservation