Quick extraction buffer

Manufactured by Illumina

The Quick extraction buffer is a laboratory reagent designed to facilitate the rapid extraction of genetic material from biological samples. It provides a simple and efficient method for isolating DNA or RNA from a variety of sources, such as tissue, cells, or environmental samples. The buffer is formulated to disrupt cellular structures and release the nucleic acids, allowing for their subsequent purification and analysis.

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Lab products found in correlation

Purelink genomic dna mini kit, by Thermo Fisher Scientific (3 mentions) Phusion flash pcr master mix, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Dna extraction kit, by Qiagen (1 mentions) I scei endonuclease, by New England Biolabs (1 mentions) Tbe gel, by Thermo Fisher Scientific (1 mentions) Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)

4 protocols using quick extraction buffer

CRISPR Genome Editing in Cell Lines

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HEK293T cells, U-2 OS cells and A549 cells acquired from ATCC were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (Gibco) and 1% (v/v) Penicillin/Streptomycin (Gibco). Cells were cultured at 37 °C with 5% CO₂.
HEK293T cells were seeded on 12-well plates overnight at 100,000 cells per well. One microgram PE2, 500 ng pegRNA, and 500 ng nicking sgRNA were transfected using Lipofectamine 3000 (Invitrogen). Cells were collected 4 days after transfection, lysed with 100 μL Quick extraction buffer (Epicenter), and incubated on a thermocycler at 65 °C for 15 min and 98 °C for 5 min. Sequences of primers used for genomic DNA amplification are listed in Supplementary Table 2.

Zheng C., Liu B., Dong X., Gaston N., Sontheimer E.J, & Xue W. (2023). Template-jumping prime editing enables large insertion and exon rewriting in vivo. Nature Communications, 14, 3369.

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Genomic DNA Extraction from Cells

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To extract the genomic DNA from HEK293T cells, cells (5 days post transfection) were washed with PBS, pelleted, lysed with 50 µl Quick extraction buffer (Epicenter), and incubated in a thermocycler (65 °C 15 min and 98 °C 5 min). 2 × 10⁵ 16HBE14o- cells (wild type, W1282X, corrected pools and three single-cell clones) were used for extracting genomic DNA using PureLink Genomic DNA Mini Kit (Thermo Fisher). The same Kit was also used to extract genomic DNA from mouse liver tissues (~ 10 mg each), three samples (from different liver lobes) per mouse.

Jiang T., Henderson J.M., Coote K., Cheng Y., Valley H.C., Zhang X.O., Wang Q., Rhym L.H., Cao Y., Newby G.A., Bihler H., Mense M., Weng Z., Anderson D.G., McCaffrey A.P., Liu D.R, & Xue W. (2020). Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope. Nature Communications, 11, 1979.

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Genomic DNA Extraction and Analysis

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To extract genomic DNA, HEK293T cells (3 days post transfection) were washed with PBS, pelleted, and lysed with 50μl Quick extraction buffer (Epicenter) and incubated in a thermocycler (65°C 15 min, and 98°C 5 min). PureLink Genomic DNA Mini Kit (Thermo Fisher) was used to extract genomic DNA from two different liver lobes (~10 mg each) per mouse. Target sequences were amplified using Phusion Flash PCR Master Mix (Thermo Fisher) with the primers listed in Supplementary Table 2. PCR products were analyzed by electrophoresis in a 1% agarose gel, and target amplicons were extracted using DNA extraction kit (Qiagen). 10 ng of purified PCR products were incubated with I-SceI endonuclease (NEB) according to manufacturer’s instruction. One-hour post incubation, the product was visualized and analyzed by electrophoresis in 4–20% TBE gel (Thermo).

Jiang T., Zhang X.O., Weng Z, & Xue W. (2021). Deletion and replacement of long genomic sequences using prime editing. Nature biotechnology, 40(2), 227-234.

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CRISPR Genome Editing in HEK293T Cells

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HEK293T and HEK293T reporter cells were seeded on 12-well plates at 100,000 cells per well. 24 hours after seeding, cells were transfected using Lipofectamine 3000 reagent (Invitrogen) following the manufacturer's protocol. Briefly, 1 μg PE2, 330 ng pegRNA, and 110 ng nicking sgRNA were transfected using 3 μL Lipofectamine 3000 and 3 μL P3000 (2 μl/μg DNA). For split-cPE2, HEK293T cells were cotransfected with 1 μg N-terminal fragment, 1 μg C-terminal fragment, 330 ng pegRNA, and 110 ng nicking sgRNA using 3 μL Lipofectamine 3000 and 5 μL P3000. For genomic DNA extraction from cells, cells were cultured for 3 days after transfection, washed with PBS, pelleted, lysed with 100 μL Quick extraction buffer (Epicenter), and incubated at 65°C for 15 min and 98°C for 5 min. To extract genomic DNA from mouse liver tissue, PureLink Genomic DNA Mini Kit (Thermo Fisher) was used following the manufacturer's protocol.

Zheng C., Liang S., Liu Y., Liu P., Kwan S., Wolfe S.A., & Xue W. (2021). Development of a flexible split prime editor using truncated reverse transcriptase.

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