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" - gRNA1/Cas9 "2" - gRNA2/Cas9 "4" - gRNA4T/Cas9n, gRNA4B/Cas9n "Blank" - mock transfection with H2O instead of plasmid Annotated map of gRNA target sites is at https://benchling.com/cshlsynbio/mammalian-crispr/tHp-puas-tk-luc/edit Extracted genomic DNA from these using the QIAmp genomic DNA mini kit (8/03/14) 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