Haynes Lab:Notebook/CRISPR Editing/2014/08/05

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08/05/14 SURVEYOR assay - for mCherry CRISPR qPCR melt curve optimization - mCherry CRISPR 1 SURVEYOR assay Assistance from Week 2 Team 1 PCR primers Forward = q1F, Reverse = P3R Amplicon length = 329 bp Annotated map at https://benchling.com/cshlsynbio/mammalian-crispr/Rc6-kah154/edit PCR templates CRISPR "1" CRISPR "2" CRISPR "4" "Blank" (3x) PCR reaction Reactions: 20 ng template DNA, 1x HF Phusion buffer, 0.2 mM dNTPs, 2.5 μL of 10 μM for each primer, 0.5 μL Phusion polymerase; in 50 μL final volume Program 98°C/ 30 sec 30x [98°C/ 10 sec, 58°C/ 15 sec, 72°C/ 15 sec] 72°C/ 10 min 4°C hold PCR product clean-up Used QIAquick PCR Purification kit Elute with 50 μL H2O Sample OD 260 260/280 ng/uL CRISPR "1" --- 1.82 40.3 CRISPR "2" --- 1.62 4.7 CRISPR "4" --- 1.85 9.8 Blank 1 --- 1.80 22.9 Blank 2 --- 1.54 5.0 Blank 3 --- 1.85 21.5 Combined Blank 1 and Blank 3, ~22 ng/μL in 100 uL volume Mismatch annealing Testing 2 methods IDT approach - mix the mutant PCR sample with a wild type reference sample to ensure heteroduplex formation Zhang Lab approach - do not mix with a wild type reference, since ~50% or more of the amplicons in the mutated cell sample will be wild type anyway 200 ng clean PCR, 1x HF Phusion buffer, in final volume of 15 μL 100 ng CRISPR "1" + 100 ng Blank (IDT approach) 200 ng CRISPR "1" (Zhang approach) 200 ng Blank (wild type control) Thermal cycler program 95ºC/ 10 min 95ºC -- (‐2.0 ºC/s) --> 85ºC 85ºC/ 1 min 85ºC -- (‐0.3 ºC/s) --> 75ºC 75ºC/ 1 min 75ºC -- (‐0.3 ºC/s) --> 65ºC 65ºC/ 1 min 65ºC -- (‐0.3 ºC/s) --> 55ºC 55ºC/ 1 min 55ºC -- (‐0.3 ºC/s) --> 45ºC 45ºC/ 1 min 45ºC -- (‐0.3 ºC/s) --> 35ºC 35ºC/ 1 min   35ºC -- (‐0.3 ºC/s) --> 25 ºC 25ºC/ 1 min 4 ºC/ hold ∞  qPCR melt curve optimization - mCherry CRISPR 1 Assistance from Kumaran Lleng Optimization parameters Borun et al reported cleaner peak profiles after a 30-cycle amplification (vs. 40 cycles) We will use 30 cycles instead of 45 Borun et al used at LEAST 50 ng genomic DNA in their reactions (final amount per PCR ambiguous) We will test 20 ng, 50 ng, and 70 ng (70 is our upper limit because of the concentration of the Blank DNA sample) Master reaction list Rxn # Template Target (primers) Method 1 1 20 ng q2F_q3R CNV melt 2 1 20 ng GAPDH B2 " 3 1 50 ng q2F_q3R " 4 1 50 ng GAPDH B2 " 5 1 70 ng P3F_q3R " 6 1 70 ng GAPDH B2 " 7 Blank 20 ng q2F_q3R " 8 Blank 20 ng GAPDH B2 " 9 Blank 50 ng q2F_q3R " 10 Blank 50 ng GAPDH B2 " 11 Blank 70 ng q2F_q3R " 12 Blank 70 ng GAPDH B2 " Notes: Opaque white plates Final reaction volumes = 15 μL Each reaction had 3.0 μL of 750 nM F/R primers, 20 ng - 70 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 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 nTc for q2F_q3R and GAPDH confirmed no background signal under identical conditions on 08/04/14 Run PCR - Bio-Rad CFX96 95°C, 3 min 30x [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec (measure)] 72°C, 30 sec Melt: 70°C -- +0.1°C/ 5 sec (measure) --> 95°C