I scei endonuclease

Manufactured by New England Biolabs

Sourced in United States

I-SceI endonuclease is a site-specific DNA endonuclease that recognizes and cleaves the 18-base pair recognition sequence 5'-TAGGGATAACAGGGTAAT-3'. It can be used for various molecular biology applications that require site-specific DNA cleavage.

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Lab products found in correlation

Phusion flash pcr master mix, by Thermo Fisher Scientific (2 mentions) Scriptcap m7g capping system, by CellScript (1 mentions) Megascript t7 transcription kit, by Thermo Fisher Scientific (1 mentions) Lipofectamine messengermax, by Thermo Fisher Scientific (1 mentions) Eclipse ti s fluorescent microscope, by Nikon (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Exfect transfection reagent, by Vazyme (1 mentions) Dna extraction kit, by Qiagen (1 mentions) Purelink genomic dna mini kit, by Thermo Fisher Scientific (1 mentions) Quick extraction buffer, by Illumina (1 mentions) Tbe gel, by Thermo Fisher Scientific (1 mentions)

3 protocols using i scei endonuclease

Production of Modified mRNA via IVT

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Template DNA was generated by linearizing the aforementioned plasmids using I-SceI endonuclease (New England Biolabs, Ipswich, MA, USA) before IVT with the MEGAscript T7 Transcription Kit (Life Technologies, Waltham, MA, USA). Post-transcriptional modifications were applied using the ScriptCap m⁷G Capping System, 2′-O-Methyltransferase Kit, and the A-Plus Poly(A) Polymerase Tailing Kit (CELLSCRIPT, Madison, WI, USA). All mRNAs were purified with the RNeasy Mini Kit (QIAGEN) after IVT and after each modification. For production of modified mRNA, the uridine nucleotide was completely replaced by N1-methylpseudouridine (tebu-bio, Boechout, Belgium) during IVT. Correct translation of EGFP-encoding IVT mRNAs and pDNA was verified after transfection of baby hamster kidney (BHK) cells using Lipofectamine MessengerMAX (Thermo Fisher Scientific) and subsequent evaluation using the Nikon Eclipse Ti-S fluorescent microscope (Nikon, Leuven, Belgium).

Huysmans H., Zhong Z., De Temmerman J., Mui B.L., Tam Y.K., Mc Cafferty S., Gitsels A., Vanrompay D, & Sanders N.N. (2019). Expression Kinetics and Innate Immune Response after Electroporation and LNP-Mediated Delivery of a Self-Amplifying mRNA in the Skin. Molecular Therapy. Nucleic Acids, 17, 867-878.

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DNA Double-Strand Break Repair Assay

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DNA DSB repair reporter plasmids were used to assess the effect of TMZ-R cells on DDR following a previously described protocol [47 ]. The system is based on an enhanced green fluorescent protein (EGFP) reporter for each type of repair. Plasmids are linearized by restriction digest with I-SceI endonuclease (R0694S, NEB, MA, USA), exposing “broken” DNA ends, and transfected into cells with ExFect transfection reagent (Vazyme Biotech, Nanjing, China) following the manufacturer’s instructions. If the repair machinery within cells is active, the linear break is repaired, reconstituting the EGFP gene and emitting a green fluorescence readout. In both instances, p-EBFP2-N1 plasmid (MiaoLing Plasmid Sharing Platform) was included as an internal transfection control.

Zhang X., Li T., Yang M., Du Q., Wang R., Fu B., Tan Y., Cao M., Chen Y., Wang Q, & Hu R. (2022). Acquired temozolomide resistance in MGMTlow gliomas is associated with regulation of homologous recombination repair by ROCK2. Cell Death & Disease, 13(2), 138.

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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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