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Acc65i

Manufactured by Thermo Fisher Scientific
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Sourced in Switzerland

The Acc65I is a lab equipment product designed for DNA restriction digestion. It functions as a type II restriction endonuclease that recognizes and cleaves the DNA sequence 'GGTACC'.

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Lab products found in correlation

Xhoi, by Thermo Fisher Scientific (2 mentions) Xbai, by Thermo Fisher Scientific (1 mentions) Nucleobond ax plasmid purification kit, by Macherey-Nagel (1 mentions) Eco32i, by Thermo Fisher Scientific (1 mentions) Sc 47778, by Santa Cruz Biotechnology (1 mentions) Sc 518025, by Santa Cruz Biotechnology (1 mentions) Lipofectamintm 2000, by Thermo Fisher Scientific (1 mentions) Accutaq la dna polymerase, by Merck Group (1 mentions) T4 polynucleotide kinase, by Thermo Fisher Scientific (1 mentions) Dual luciferase reporter 1000 assay system, by Promega (1 mentions) Bigdye terminator, by Thermo Fisher Scientific (1 mentions) Taq polymerase, by Thermo Fisher Scientific (1 mentions) Protein assay, by Bio-Rad (1 mentions) Qiaquick gel extraction kit, by Qiagen (1 mentions) T4 dna ligase, by Promega (1 mentions) Nucleospin tissue, by Macherey-Nagel (1 mentions) Pcibn deltanls pmgfp, by Addgene (1 mentions) Eco47iii, by Thermo Fisher Scientific (1 mentions) Hieff clone plus one step cloning kit, by Yeasen (1 mentions) Long pcr enzyme mix, by Thermo Fisher Scientific (1 mentions)

4 protocols using acc65i

1

Verification of Silent Mutations

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In order to verify deposition of Silent mutations following excision with unilaterally or bilaterally mutant µH, genomic DNA was amplified using primer set dna1720/411 (HPRT1) or dna1711/1712 (APRT). Cleaved amplicons were resolved by gel electrophoresis following treatment with or without AflII (HPRT1) or Acc65I (APRT) restriction enzyme (Fermentas).
Kim S.I., Matsumoto T., Kagawa H., Nakamura M., Hirohata R., Ueno A., Ohishi M., Sakuma T., Soga T., Yamamoto T, & Woltjen K. (2018). Microhomology-assisted scarless genome editing in human iPSCs. Nature Communications, 9, 939.
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2

Molecular Cloning and Protein Expression Techniques

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AccuTaq LA DNA Polymerase was from Sigma (Sigma-Aldrich, St. Louis, MO, USA), the T4 DNA Ligase from Promega (Promega, Switzerland), T4 Polynucleotide Kinase, Shrimp Alkaline Phosphatese, restriction enzymes Acc65I, AccI, SmaI, Hind II, XhoI, NheI, Cfr9I, XbaI, XmaJi, Cfr10I, Eco32I from Fermentas (Fermentas, Payerne, Switzerland), Taq-Polymerase and LipofectaminTM 2000 from Invitrogen (Invitrogen, Switzerland), PfuUltra Hotstar DNA Polymerase from Agilent (Agilent, Basel, Switzerland), and the Big dye Terminator from Applied Biosystems (Applied Biosystems, Muttenz, Switzerland). The p-38 MAPK inhibitor (SB203580) was obtained from Jena Bioscience (Jena Bioscience GmbH, Jena, Germany). Antibodies against β-actin (sc-47778, 1:1000), PGC-1α (sc-518025, 1:1000), and MEF2 (1:1000) were from Santa Cruz Biotechnology (Dallas, TX, USA), against OCTN2 (ab180757, 1:1000) from Abcam (Cambridge, UK) and the control IgG from Santa Cruz Biotechnology (Dallas, TX, USA).
The DNA purification kit NucleoSpin®Tissue and the NucleoBond®AX Plasmid Purification kit were from Macherey-Nagel (Düren, Germany), the QIAquick Gel Extraction Kit from Qiagen (Hilden, Germany), the Dual-Luciferase® Reporter 1000 Assay System from Promega (Promega, Switzerland), and Micro Bio-spin chromatography columns and the Bio-Rad protein assay from Bio-Rad (Bio-Rad Laboratories AG, Cressier, Switzerland).
Novakova K., Török M., Panajatovic M., Bouitbir J., Duong F.H., Handschin C, & Krähenbühl S. (2022). PGC-1α and MEF2 Regulate the Transcription of the Carnitine Transporter OCTN2 Gene in C2C12 Cells and in Mouse Skeletal Muscle. International Journal of Molecular Sciences, 23(20), 12304.
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3

Mammalian Luminescence Biosensor Systems

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For mammalian expression of the LuminON system, the coding sequence of Nanoluc was cloned into pGAVPO and pSV40-GAVPO27 (link) using the Hieff Clone® Plus One Step Cloning Kit (YEASEN) to obtain plasmids expressing different configurations of Nluc-GAVPO fusion proteins driven by the CMV or SV40 promoter. For application of the LOVTRAP or CRY2-CIB1 system, Nanoluc was ligated into the pTriEx-mCherry-LOV2 (Addgene: 81041) plasmid digested by Acc65I and NheI (Thermo Scientific), the pCIBN(deltaNLS)-pmGFP (Addgene: 26867) plasmid digested by Eco47III and AgeI (Thermo Scientific), or the pCRY2FL(deltaNLS)-mCherryN1 (Addgene: 26871) plasmid digested by Eco47III and XhoI (Thermo Scientific). For cell therapy of the LuminON system in mice, luminGAVPO was ligated into the pYH88 plasmid digested by Eco47III and BsrGI, and UASG-TATA-Gluc-P2A-mINS was ligated into the pWS251 (obtained from Haifeng Ye, ECNU) plasmid digested by NotI and MluI.
Li T., Chen X., Qian Y., Shao J., Li X., Liu S., Zhu L., Zhao Y., Ye H, & Yang Y. (2021). A synthetic BRET-based optogenetic device for pulsatile transgene expression enabling glucose homeostasis in mice. Nature Communications, 12, 615.
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4

Inverse PCR for Identifying Locus Target

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For the identification of the locus that was targeted by integration of the amdS marker in QM9414_Dhax1 strains, an inverse PCR was performed as follows. 20 µg of chromosomal DNA were digested with either Acc65I or NotI (Thermo Scientific) at a final concentration of 1 U/µl in a total reaction volume of 20 µl according to the manufacturer’s instructions. After heat inactivation, 2 µl of T4 Ligase (Promega, 1–3 U/µl) and the corresponding buffer were added and ligation was performed at 18 °C for 90 min. Subsequently, the ligation was stopped by heat inactivation and 1 µl was applied as template in a 25 µl inverse PCR reaction, initially using the primers amdS inv for and amdS inv rev. For further approaches, the primers locus for and locus rev annealing to the identified regions were applied. All amplifications were performed in an iCycler (Bio-Rad, Hercules, CA, USA) using the Long-PCR Enzyme Mix (Thermo Scientific) and the following program: initial denaturation at 94 °C for 3 min, followed by 30 cycles of 30 s at 94 °C, 30 s at 59 °C and 3 min at 72 °C, and final elongation at 72 °C for 5 min. The DNA fragments were sequenced by MWG Biotech (Ebersberg, Germany).
Till P., Pucher M.E., Mach R.L, & Mach-Aigner A.R. (2018). A long noncoding RNA promotes cellulase expression in Trichoderma reesei. Biotechnology for Biofuels, 11, 78.
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