Genomic DNA was extracted from cell pools using QIAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany) and from monoclonal cell populations using Quick Extract™ DNA Extraction Solution (Lucigen, Middleton, WI, USA), according to the manufacturer’s instructions. Plasmid DNA from bacterial mini cultures was extracted using alkaline lysis as described elsewhere [107 (link)]. Larger-scale plasmid preparations were isolated by silica-based plasmid DNA purification using the NucleoBond® Xtra Midi Kit and Maxi Kit (Macherey Nagel, Düren, Germany), according to the manufacturer’s instructions. Cycle sequencing reactions were prepared using the BigDye Terminator v1.1 Cycler sequencing kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions, and analyzed on a Hitachi 3031xl Genetic Analyzer with Sequence Detection Software version 5.2 (Applied Biosystems, Foster City, CA, USA).
Bigdye terminator v1.1 cycler sequencing kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BigDye Terminator v1.1 Cycler sequencing kit is a DNA sequencing reagent system used to perform DNA sequencing analysis. The kit contains reagents required for the Sanger sequencing method, including DNA polymerase, fluorescently labeled dideoxynucleotides, and other necessary components.
Automatically generated - may contain errors
Lab products found in correlation
Qiamp dna blood mini kit, by Qiagen (3 mentions)
Nucleobond xtra midi kit, by Macherey-Nagel (1 mentions)
Maxi kit, by Macherey-Nagel (1 mentions)
Gc rich solution, by Roche (1 mentions)
Veriti thermal cycler, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Q5 hot start high fidelity dna polymerase, by New England Biolabs (1 mentions)
4 protocols using bigdye terminator v1.1 cycler sequencing kit
1
Genomic and Plasmid DNA Extraction and Sequencing
2
Automated DNA Sequencing Protocol
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Purified plasmids and PCR products were sequenced using the BigDye Terminator v1.1 Cycler sequencing kit (Applied Biosystems, Foster City, CA, USA), including 1 × GC-RICH solution (Roche, Basel, Switzerland). DNA sequencing products were purified using Performa® DTR Gel Filtration Cartridges Performa® DTR Gel Filtration Cartridges (Edge Biosystems, Gaithersburg, MD, USA, USA) and analyzed on a Hitachi 3031xl Genetic Analyzer with the Sequence Detection Software version 5.2 (Applied Biosystems, Foster City, MA, USA).
3
Eukaryotic Genomic DNA Extraction and Sequencing
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Genomic DNA from eukaryotic cells was extracted using QIAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Cycle sequencing was performed using the BigDye Terminator v.1.1 Cycler sequencing kit (Applied Biosystems, Waltham, Massachusetts, USA) and the Veriti thermal cycler (TFS). Performa DTR Gel Filtration Cartridges (Edge Biosystems, Gaithersburg, Maryland, USA) were used for purification of sequencing products, all in accordance with manufacturers’ instructions, and analyzed on a Hitachi 3031xl Genetic Analyzer with Sequence Detection Software v.5.2 (Applied Biosystems).
4
DNA Editing Analysis Protocol
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Five to seven days post-nucleofection, approximately 3 × 105 cells were collected for DNA analysis. The genomic DNA was extracted from cells using the QIAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, and was amplified by polymerase chain reaction (PCR) using Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, United States). The PCR product was purified using the QIAquick® PCR Purification Kit (Qiagen Hilden, Germany) and was then processed for Sanger sequencing (for primer sequences see Supplementary Table S3 ) as the basis for the assessment of DNA editing. For cycle sequencing, the BigDye™ Terminator v1.1 Cycler Sequencing Kit (Applied Biosystems, Waltham, MA, United States) was used according to the manufacturer’s instructions.
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