DNeasy Blood & Tissue Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Downstream tips
- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Publication protocol

Detection of CRISPR/Cas9-facilitated excision
HEK293 and C2C12 cells were transfected using FuGene HD (Promega, Madison, WI). Briefly, 150 000 cells were plated 1–2 days prior on poly--lysine-coated (HEK293) or standard (C2C12) 24-well tissue culture plates and were transfected with 5 μg (HEK293) or 3 μg (C2C12) of each sgRNA/Cas9 vector for a total of 10 or 6 μg per transfection, respectively, in Opti-MEM I (Life Technologies) medium with a 3:1 ratio of FuGene HD:plasmid DNA. Cells were harvested 4 days post-transfection and genomic DNA was isolated using the DNeasy Blood and Tissue Mini Kit (Qiagen). Each transfection was performed at least twice. H9 hESCs were transfected using the Amaxa Nucleofector (Lonza, Basel, Switzerland) with the Human Embryonic Stem Cell Nucleofection Kit 2 (Lonza). Briefly, cells were passaged normally into Matrigel-coated 6-well plates three days prior to nucleofection. Before nucleofection, medium was replaced at least 1 h beforehand with fresh medium containing 10 μM ROCK inhibitor Y-27632 (R&D Systems, Minneapolis, MN). Cells were dissociated with collagenase IV and subsequently electroporated with 1.5 μg of each sgRNA/vector for a total of 3 μg using program B-16 according to the manufacturer's instructions, and plated on Matrigel-coated 12-well plates in human stem cell medium with 10 μM ROCK inhibitor Y-27632. Each reaction was carried out at least twice Genomic DNA was isolated from the cells 2 days later with the DNeasy Blood and Tissue Mini Kit.

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Manufacturer protocol

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