Quickextract solution

Manufactured by Illumina

Sourced in United States

The QuickExtract solution is a DNA extraction kit designed for rapid and efficient DNA isolation from a variety of sample types, including tissues, cells, and microorganisms. The solution utilizes a proprietary extraction method to release DNA from samples, which can then be used in downstream applications such as PCR and sequencing.

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Lab products found in correlation

27 protocols using quickextract solution

Detecting CRISPR-Cas9 Genome Editing at Rosa26 Locus

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The activity of gRNAs targeting the Rosa26 locus were initially tested by induction of NHEJ. Cells transfected with Cas9-RNP targeting the Rosa26 locus were harvested 4 days after electroporation, washed once in PBS and genomic DNA was recovered from 1 × 10⁶ cells using 100 μl Quick Extract solution (Epicenter) according to the manufacturer's instructions. Small fragments of DNA covering the putative cleavage sites were amplified by PCR with KAPA Hifi HotStart Ready Mix (KAPA Biosystems, KK2602) from the genomic DNA using primers p13 and p14. Control DNA was also amplified from wildtype C57BL/6-Ly5.1 genomic DNA. CRISPR-Cas9 cleavage of the genome was determined using a Surveyor Mutation Detection Kit (IDT, 706020). All samples were run on 2% gels for the detection of cleavage products. For reference, GeneRuler 1 kb DNA Ladder (Thermo, SM0314) and GeneRuler 100 bp DNA Ladder (Thermo, SM0243) were used as DNA size markers.

Pesch T., Bonati L., Kelton W., Parola C., Ehling R.A., Csepregi L., Kitamura D, & Reddy S.T. (2019). Molecular Design, Optimization, and Genomic Integration of Chimeric B Cell Receptors in Murine B Cells. Frontiers in Immunology, 10, 2630.

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Cas9 RNP Preparation and Cell Lysis

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The Cas9 RNP was prepared according the protocols described previously (42 (link)). The Cas9 RNP was made immediately before the experiment. To make the Cas9 RNP, purified Cas9 protein was incubated with sgRNA at a 1:1.2 molar ratio in 20 μM Hepes at pH 7.5, 150 mM KCl, 1 mM MgCl₂, 10% glycerol, and 1 mM TCEP at 37°C for 10 min. Cells were incubated at 37°C for 24 to 48 hours after Cas9 RNP delivery. To extract the genomic DNA, the cells were lysed by 20 to 100 μl of QuickExtract solution (Epicentre) at 65°C for 20 min and then at 95°C for 20 min and were stored at −20°C. The concentration of genomic DNA was measured by NanoDrop (Thermo Scientific).

Cao Y., Chen H., Qiu R., Hanna M., Ma E., Hjort M., Zhang A., Lewis R.S., Wu J.C, & Melosh N.A. (2018). Universal intracellular biomolecule delivery with precise dosage control. Science Advances, 4(10), eaat8131.

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Cloning and Sequencing of H2-K Locus

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Unless otherwise specified all primers were ordered from Integrated DNA Technologies (IDT). Genomic DNA was extracted from RAW264.7 and JAWSII cells (1 × 10⁶ each) by the addition of 100 μl QuickExtract solution (Epicentre) according to the manufacturers instructions. PCR using the Kapa2G Fast Readymix (Kapa Biosystems) and with primers p11 and p12 was performed to amplify ~4300 bp of DNA at the H2-K locus. Primers p13 and p14 were used to amplify a backbone fragment from pUC19 plasmid [New England Biolabs (NEB)]. Gibson cloning master mix (NEB) was used to combine the respective fragments to create the storage plasmids pUC19-H2-Kb and pUC19-H2-Kd, later used for Sanger sequencing. Additionally, ~1 kb regions 5′ and 3′ to the H2-Kd locus were amplified using primers p15-p16 and p17-p18, respectively and Sanger sequenced.

Kelton W., Waindok A.C., Pesch T., Pogson M., Ford K., Parola C, & Reddy S.T. (2017). Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange. Scientific Reports, 7, 45775.

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Genotyping Hoxb5-tri-mCherry Mice

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Genomic DNA from Hoxb5-tri-mCherry mice was isolated from tail biopsies using QuickExtract solution (EpiCentre). PCR amplification was performed using the same forward primer (F: 5’-GACGTATCGAGATCGCCCAC-3’) with two reverse primers to distinguish between the Hoxb5-tri-mCherry (R1: 5’-CCTTGGTCACCTTCAGCTTGG-3’) and wild-type (R2: 5’-AGATTGGAAGGGTCGAGCTG-3’) alleles.

Chen J.Y., Miyanishi M., Wang S.K., Yamazaki S., Sinha R., Kao K.S., Nakauchi H, & Weissman I.L. (2016). Hoxb5 marks long-term haematopoietic stem cells revealing a homogenous perivascular niche. Nature, 530(7589), 223-227.

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Genotyping of SERPINE1-FOSB Fusion

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PCR screening was performed to determine the presence of both the 5′ homology arm of SERPINE1 (primers SF and FR), the 3′ homology arm of FOSB (primer F2 and R2), the wild-type SERPINE1 (primer SF and SR) and wild-type FOSB (primer FF and FR) in clonal lines (Table S1). Colonies were picked in maximum 2 μl hESC-food and added to 20 μl QuickExtract Solution (Epicenter) in 0.5 mL tubes. The tubes were vortexed for 15 s and DNA was extracted by heating the samples to 65°C for 15 minutes, 68°C for 15 minutes and 98°C for 10 minutes in a thermocycler. 2-Step PCR was performed with Terra PCR Direct Polymerase (TaKaRa) according to the manufacturer’s protocol. Sanger sequencing was performed (BaseClear) to confirm the SERPINE1-FOSB fusion and to screen the SERPINE1 and FOSB wild-type allele for on-target mutations due to NHEJ.

van IJzendoorn D.G., Salvatori D.C., Cao X., van den Hil F., Briaire-de Bruijn I.H., de Jong D., Mei H., Mummery C.L., Szuhai K., Bovée J.V, & Orlova V.V. (2020). Vascular Tumor Recapitulated in Endothelial Cells from hiPSCs Engineered to Express the SERPINE1-FOSB Translocation. Cell Reports Medicine, 1(9), 100153.

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Genomic DNA Isolation from iPSCs

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To isolate genomic DNA from iPSCs, 30 μl Quick Extract Solution (Epicentre, Madison, WI, United States) was added to each cell pellet and incubated for 15 min at 65°C, followed by 5 min at 95°C. PCR was carried out using gene-specific primers (Table 1), and 2 μl of PCR product were used for further allele-specific SNP genotyping with Quantstudio7 (Thermo Fisher Scientific, Waltham, MA, United States). After confirmation of gene edit, ∼100 ng of PCR product was treated with Exosap-IT (Thermo Fisher Scientific, Waltham, MA, United States) and sent to Retrogen (San Diego, CA, United States) for Sanger sequencing. Clones that were positive for iPSCs containing the edited MYH6-R443P gene were sequenced at the conserved region in MYH7 as well to check the CRISPR’s off-target activity.

Kim M.S., Fleres B., Lovett J., Anfinson M., Samudrala S.S., Kelly L.J., Teigen L.E., Cavanaugh M., Marquez M., Geurts A.M., Lough J.W., Mitchell M.E., Fitts R.H, & Tomita-Mitchell A. (2020). Contractility of Induced Pluripotent Stem Cell-Cardiomyocytes With an MYH6 Head Domain Variant Associated With Hypoplastic Left Heart Syndrome. Frontiers in Cell and Developmental Biology, 8, 440.

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CRISPR-Mediated Genome Editing in HEK293T Cells

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HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, and 10% fetal bovine serum. sgRNA plasmid (500 ng) and effector [nuclease, BE3, or ABE(7.10)] plasmid (500 ng) were transfected into cells as duplicates (2 × 10⁵ per well in a 24-well plate) with Lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco). After 48 hours posttransfection, genomic DNA was extracted using QuickExtract Solution (Epicentre), and genomic loci were amplified by PCR using the KAPA HiFi HotStart ReadyMix (Kapa Biosystems). For base-editing analysis, amplicons were purified and submitted for Sanger sequencing (Genewiz). For indel analysis, the T7E1 reaction was conducted according to the manufacturer’s instructions, and equal volumes of products were analyzed on a 2% agarose gel stained with SYBR Safe (Thermo Fisher Scientific). Unprocessed gel image files were analyzed in Fiji (41 (link)). The cleaved bands of interest were isolated using the rectangle tool, and the areas under the corresponding peaks were measured and calculated as the fraction cleaved of the total product. Percent gene modification was calculated as follows (42 (link))

% gene modification = 100 \times (1 - {(1 - fraction cleaved)}^{\frac{1}{2}})

All samples were performed in duplicates, and percent gene modifications were averaged. SD was used to calculate error bars.

Chatterjee P., Jakimo N, & Jacobson J.M. (2018). Minimal PAM specificity of a highly similar SpCas9 ortholog. Science Advances, 4(10), eaau0766.

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Efficient Cas9 RNP Cutting Evaluation

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To select optimal gRNAs, genomic cutting using Cas9 RNPs was estimated using T7 endonuclease I assay. Genomic DNA was extracted from edited cells (HUDEP-2 or CD34+ HSPC) using QuickExtract solution (Epicentre, Inc.) to a density of >2,500 cells/μL. A PCR amplicon of the targeted region was generated using GXL Primestar Polymerase (Takara, Inc.), 30 PCR cycles, and manufacturer’s instructions, at a final volume of 50 μL using 5 μL of extracted genomic DNA as template. 7 μL of unpurified PCR was mixed with water and 1X NEB buffer 2 to a final volume of 10 μL before re-annealing in a thermal cycler (95 degrees for 5 minutes, cool to room temperature over 10 minutes). 1 μL of T7 endonuclease I was added (NEB, Inc.) and reaction was digested for 30 minutes at 37°C before visualization on a 2% agarose gel or 4–20% polyacrylamide TBE gel (Life Technologies, Inc.). Cutting was compared to PCR amplicons from untreated cells.

Chung J.E., Magis W., Vu J., Heo S.J., Wartiovaara K., Walters M.C., Kurita R., Nakamura Y., Boffelli D., Martin D.I., Corn J.E, & DeWitt M.A. (2019). CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells. PLoS ONE, 14(1), e0208237.

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MAOA-uVNTR Genotyping Protocol

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DNA was extracted from buccal swab samples, using the QuickExtract solution and protocol from Epicentre (Madison, WI). MAOA-uVNTR allelic variants were genotyped using PCR-based amplification, with the following primers: forward, 5’- ACAGCCTGACCGTGGAGAAG-3’; and reverse, 5’-GAACGGACGCTCCATTCGGA-3’. PCR reactions contained 50 ng of template DNA, 1.0 U GoTaq Flexi DNA polymerase (Promega), 1.0 µM of each primer, 0.3 mM dNTP, 2.0 mM MgCl2, and 5 µl of 5× Green Reaction Buffer (Promega) in a total volume of 25 µl. volume. After 2 min at 95°C, 35 cycles were carried out at 95°C for 1 min, at 59°C for 1 min, and at 72°C for 1 min, with a final extension at 72°C for 5 min. PCR products were separated on 3% agarose gels and visualized by ethidium bromide staining. All laboratory procedures were carried out blind to the case-control status. All participants were found to harbor 3-repeat (L-MAOA) or 4-repeat variants (H-MAOA), with the exception of one carrier of the 2-repeat variant, who was added to the L-MAOA group (Sabol et al. 1998 (link)) for all statistical analyses.

Stetler D.A., Davis C., Leavitt K., Schriger I., Benson K., Bhakta S., Wang L.C., Oben C., Watters M., Haghnegahdar T, & Bortolato M. (2014). Association of low-activity MAOA allelic variants with violent crime in incarcerated offenders. Journal of psychiatric research, 58, 69-75.

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CRISPR-Cas9 gene editing in iPSCs

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CRISPR guide RNA (gRNA) sequences were designed using online tool (http://crispr.mit.edu/), and we selected the gRNAs with highest score (specificity). The gRNAs were cloned into pSpCas9(BB)-2A-Puro vector (Addgene) for co-expression with Cas9 based on an established protocol (Ran et al., 2013 (link)). For transfection, we used FuGENE HD (Promega) at DNA/Reagent ratio of 1:3.5 to co-transfect CRISPRs (1.5 μg of Cas9-gRNA plasmid) and ssODNs (1.5 μg repair oligos) into iPSCs cultured on 6-well plate (3 × 10⁵ cells plated one day before transfection in mTeSR1 media with 5 μM ROCK inhibitor). We started puromycin (0.4 μg/ml) selection at 24 hr post transfection, and cut the puromycin to 0.2 μg/ml at 48 hr post transfection and withdrawn it after 72 hr of transfection. 10 days after transfection, resistant colonies were picked into 96-well plates and a small proportion of cells from each colony were used for DNA isolation using QuickExtract solution (Epicenter). The extracted DNAs were used for Sanger sequencing to verify the on-target and off-target editing. For checking off-target editing, primers were designed to amplify regions corresponding to the 6 top ranking predicted off-targets. Primers and oligos were synthesized at Integrated DNA Technologies (IDT).

Forrest M.P., Zhang H., Moy W., McGowan H., Leites C., Dionisio L.E., Xu Z., Shi J., Sanders A.R., Greenleaf W.J., Cowan C.A., Pang Z.P., Gejman P.V., Penzes P, & Duan J. (2017). Open Chromatin Profiling in hiPSC-Derived Neurons Prioritizes Functional Noncoding Psychiatric Risk Variants and Highlights Neurodevelopmental Loci. Cell stem cell, 21(3), 305-318.e8.

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