Restriction endonucleases

Manufactured by Roche

Sourced in Germany

Restriction endonucleases are enzymes that recognize and cleave specific DNA sequences, known as restriction sites. They are used in molecular biology and genetic engineering to manipulate DNA molecules.

Automatically generated - may contain errors

Lab products found in correlation

T4 dna ligase, by Roche (5 mentions) Qiaprep spin miniprep kit, by Qiagen (5 mentions) Standard taq polymerase, by New England Biolabs (3 mentions) Purelink genomic dna mini kit, by Thermo Fisher Scientific (3 mentions) Dimethyl sulfoxide (dmso), by Merck Group (3 mentions) Betaine, by Merck Group (3 mentions) Qiaquick pcr purification and gel extraction kits, by Qiagen (1 mentions) Expand long template kit, by Roche (1 mentions) Taq polymerase, by Roche (1 mentions) Alkaline phosphatase, by Roche (1 mentions) Ivis lumina 2 system, by PerkinElmer (1 mentions) Isopropyl β d thiogalactopyranoside iptg, by Roche (1 mentions) E coli bl21 de3 cells, by Promega (1 mentions) Nickel nitrilotriacetic acid ni nta agarose, by Qiagen (1 mentions) High fidelity dna polymerase, by Roche (1 mentions) Prestained molecular weight markers, by Bio-Rad (1 mentions) Thiamine hcl, by Merck Group (1 mentions) Bis acrylamide, by Bio-Rad (1 mentions) Acrylamide, by Bio-Rad (1 mentions) Ammonium persulfate, by Bio-Rad (1 mentions)

9 protocols using restriction endonucleases

Molecular Cloning Techniques in Lactobacillus

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DNA isolation, endonuclease treatment and ligation, were performed as previously described (Fiocco et al., 2009 (link)). Taq polymerases, restriction endonucleases, alkaline phosphatase, and T4 DNA ligase were purchased from Roche (Milan, Italy), Invitrogen (Milan, Italy), New England Biolabs (Hertfordshire, United Kingdom), Fermentas (Burlington, ON, Canada), and Promega (Milan, Italy) and were used as recommended by the suppliers.
Plasmids were isolated using QIAprep spin miniprep kits (Qiagen, Hilden, Germany). PCR products and DNA restriction fragments were purified with the QIAquick PCR purification and gel extraction kits (Qiagen). L. plantarum genomic DNA was isolated using a microbial DNA extraction kit (Cabru, Milan, Italy) according to the manufacturer’s procedure. L. plantarum genomic DNA (approximately 20 ng) was PCR amplified using Expand Long Template kit (Roche) following the manufacturer’s instructions.

Arena M.P., Capozzi V., Longo A., Russo P., Weidmann S., Rieu A., Guzzo J., Spano G, & Fiocco D. (2019). The Phenotypic Analysis of Lactobacillus plantarum shsp Mutants Reveals a Potential Role for hsp1 in Cryotolerance. Frontiers in Microbiology, 10, 838.

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Bioluminescent Listeria Strain Generation

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CBR was codon-optimized (CBR^opt) for expression in Listeria and synthesized by GenScript (GenScript United States Inc., Piscataway, NJ, United States). The CBR^opt gene (Gene Accession No. KY628997) was synthesized with an intact upstream constitutive Listerial promoter, highly expressed Listerial promoter (P_HELP) (Riedel et al., 2007 (link)) and the fragment was sub-cloned into the pPL2 backbone (Lauer et al., 2002 (link)) in E. coli using Xho1 and Pst1 restriction enzymes resulting in pPL2CBR^opt (see Figure 1). Plasmid DNA was isolated from E. coli using a QIAprep Spin Miniprep kit according to the manufacturer’s instructions (QIAGEN, Crawley, United Kingdom). The sequences were verified by sequencing through MWG Biotech AG (Ebersberg, Germany). Transformation of L. monocytogenes was performed as described (Park and Stewart, 1990 (link)). Successful integrants were screened for site-specific integration by PCR using template DNA of L. monocytogenes as described previously (Bron et al., 2006 (link)). Restriction endonucleases (Roche Diagnostic, Mannheim, Germany), T4 DNA ligase (Roche) and 2× PCR mixture (NEB, United Kingdom) were used as advised by the manufacturer. From each successful transformant one colony carrying the anticipated genotype was chosen for further bioluminescence analysis using an IVIS Lumina II system (PerkinElmer).

Ur Rahman S., Stanton M., Casey P.G., Spagnuolo A., Bensi G., Hill C., Francis K.P., Tangney M, & Gahan C.G. (2017). Development of a Click Beetle Luciferase Reporter System for Enhanced Bioluminescence Imaging of Listeria monocytogenes: Analysis in Cell Culture and Murine Infection Models. Frontiers in Microbiology, 8, 1797.

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DNA Isolation and Sequencing Protocol

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DNA isolation was performed using the PureLink Genomic DNA mini kit (Life Technologies) except for TraDIS library genomic DNA isolation (see below). Isolation of plasmid DNA was carried out using the QIAprep spin miniprep kit (Qiagen). Primers (Sigma) used are shown in Supplementary Table 3. DNA fragments were amplified with either KOD Hot Start DNA Polymerase (Novagen) or standard Taq polymerase (NEB) as described by the manufacturer, with the inclusion of Betaine (Sigma) or DMSO (Sigma). Restriction endonucleases (Roche) were used according to the manufacturer’s specifications. DNA sequencing was performed by GATC Biotech.

Nolan L.M., Cain A.K., Clamens T., Furniss R.C., Manoli E., Sainz-Polo M.A., Dougan G., Albesa-Jové D., Parkhill J., Mavridou D.A, & Filloux A. (2021). Identification of Tse8 as a Type VI secretion system toxin from Pseudomonas aeruginosa that targets the bacterial transamidosome to inhibit protein synthesis in prey cells. Nature microbiology, 6(9), 1199-1210.

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Engineered HEV ORF2 Capsid Mutants

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Plasmid pET-28a (+)/p179 containing the 439–617aa region of HEV ORF2 of genotype 4 HEV strain has been constructed previously in our laboratory^{14 (link),28 (link)}. The p179 mutants with wild-type asparagine (N) replaced by the cyclic aa (P) and aromatic aa (Y) at position 562 were previously designed and successfully expressed in P. pastoris^{14 (link)}. HEV capsid protein-specific mAb (1G10) was produced by our research group^{13 (link)}. Isopropyl-β-D-thiogalactopyranoside (IPTG), High-fidelity DNA polymerase, dNTP, T4 DNA ligase and restriction endonucleases were purchased from Roche (Germany). E. coli BL21(DE3) cells were purchased from Promega. HRP-conjugated goat anti-mouse was from KPL (Gaithersburg, MD, USA). Trypsin and DAB were purchased from Sigma–Aldrich (St. Louis, MO, USA). Plasmid and DNA recovery/purification kits were obtained from Axygen, Inc (USA). The nickel-nitrilotriacetic acid (Ni-NTA) Agarose was obtained from QIAGEN Sciences, MD, USA.

Wei W., Behloul N., Baha S., Liu Z., Aslam M.S, & Meng J. (2018). Dimerization: a structural feature for the protection of hepatitis E virus capsid protein against trypsinization. Scientific Reports, 8, 1738.

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Recombinant LTB and CTB Protein Production

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Murashige and Skoog (MS) medium, spectinomycin, and sucrose were purchased from MB cell (CA, U.S.A). Tetracycline, cefotaxime and phosphinothricin were purchased from Duchefa (Haarlem, Netherlands). The pMJ103 vector was constructed by GreenGene BioTech (Yongin, Korea) and the plasmids (MYO51, MYO53) containing LTB and/or CTB were kindly donated from Dr. M.S. Yang (Chonbuk National University). T4 DNA ligase and restriction endonucleases were obtained from Roche (Basel, Switzerland). Gateway BP and LR recombinase systems were purchased from Invitrogen (Breda, Netherlands). A mini trans-blot cell and Mini-Protean III cell were purchased from Bio-Rad (Hercules, CA, USA). Reagents for SDS-polyacrylamide electrophoresis, such as acrylamide, bis-acrylamide, ammonium persulfate, TEMED, prestained molecular weight markers and Coomassie Brilliant Blue R-250 solution were obtained from Bio-Rad (Hercules, CA, USA). Anti-LTB polyclonal antibody was obtained from Abcam (Cambridge, MA, USA). G_M1-ganglioside and anti-rabbit IgG-conjugated alkaline phosphatase were purchased from Santa-Cruz Biotechnology (CA, USA). Thiamine-HCl, myo-inositol, anti-CTB polyclonal antibody, and other reagents such as salts and buffer components were of analytical grade and purchased from Sigma (St. Louis, MO, USA).

Soh H.S., Chung H.Y., Lee H.H., Ajjappala H., Jang K., Park J.H., Sim J.S., Lee G.Y., Lee H.J., Han Y.H., Lim J.W., Choi I., Chung I.S, & Hahn B.S. (2015). Expression and functional validation of heat-labile enterotoxin B (LTB) and cholera toxin B (CTB) subunits in transgenic rice (Oryza sativa). SpringerPlus, 4, 148.

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DNA Isolation and Sequencing Protocol

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DNA Isolation and Amplification Protocol

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DNA isolation was performed using the PureLink Genomic DNA mini kit (Life Technologies) except for TraDIS library genomic DNA isolation (see below). Isolation of plasmid DNA was carried out using the QIAprep spin miniprep kit (Qiagen). Primers (Sigma) used are shown in Table 1. DNA fragments were amplified with either KOD Hot Start DNA Polymerase (Novagen) or standard Taq polymerase (NEB) as described by the manufacturer with the inclusion of Betaine (Sigma) or DMSO (Sigma). Restriction endonucleases were used according to the manufacturer’s specifications (Roche). DNA sequencing was performed by GATC Biotech.

Nolan L.M., Whitchurch C.B., Barquist L., Katrib M., Boinett C.J., Mayho M., Goulding D., Charles I.G., Filloux A., Parkhill J, & Cain A.K. (2018). A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa. Microbial Genomics, 4(11), e000229.

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DNA Extraction and Cloning in Rhodococcus aetherivorans

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Total DNA from Rhodococcus aetherivorans BCP1 and the transposon mutants was extracted from cells grown in 50 mL liquid LB cultures incubated at 30°C for 48 h (up to OD₆₀₀ of 1–1.2). After cell harvesting, BCP1 WT and mutant cells were lysed using the protocol reported by Cappelletti et al. (2011 (link)), while standard molecular techniques were used for DNA manipulation and cloning (Sambrook et al., 1989 ). Shortly, for molecular cloning 1.5–3 μg of DNA were digested with appropriate restriction endonucleases (1 U) (Roche) for 3–4 h at 37°C. In multi‐enzyme digestion, compatible reaction buffers were used. Ligation reactions were performed overnight in a final volume of 15 μL using the T4 ligase (Roche) based on the manufacturer instructions except for the use of a temperature gradient ranging from 37°C to 4°C. Recombinant plasmids were introduced into E. coli DH5α using chemical transformation standard protocol (Sambrook et al., 1989 ). PCR and plasmid purifications were carried out using the Plasmid Mini Kit and PCR purification kit (Qiagen) according to the manufacturer's instructions. QIAquick Gel Extraction Kit (Qiagen) was used for the recovery and purification of DNA fragments excised from agarose gel.

Ferrari E., Di Benedetto G., Firrincieli A., Presentato A., Frascari D, & Cappelletti M. (2024). Unravelling the role of the group 6 soluble di‐iron monooxygenase (SDIMO) SmoABCD in alkane metabolism and chlorinated alkane degradation. Microbial Biotechnology, 17(5), e14453.

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Chromosomal DNA Isolation and Plasmid Manipulation in Bifidobacterium

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Chromosomal DNA was isolated from B. breve JCM7017‐199, a B. breve JCM7017‐derivative strain containing a tetracycline resistance marker disrupting the open reading with locus tag pMP7017_199 of plasmid pMP7017, as previously described (Bottacini et al.,2015 (link)). Plasmid DNA from E. coli was extracted using the High Pure Plasmid Isolation Kit (Sigma‐Aldrich, Gillingham, UK). Standard oligonucleotides (Table S2) used in this study were synthesized by Eurofins (Ebersberg, Germany). Both standard and colony PCRs were performed using DreamTaq PCR 2× Master Mix (Thermo Fisher Scientific, Gloucester, United Kingdom), and colony PCRs were carried out as described previously (Mazé et al.,2007 (link)). High‐fidelity PCR was achieved using Q5 High‐Fidelity 2X Master Mix (New England Biolabs, Hitchin, United Kingdom). PCR fragments were purified using the High Pure PCR Product Purification Kit (Sigma). Restriction endonucleases and T4 DNA ligase were used according to the manufacturer’s instructions (Roche, Mannheim, Germany). Electroporation of plasmid DNA into Bifidobacterium spp. was conducted as previously described (Rossi et al.,1997 ), while E. coli transformations were performed as described by Sambrook et al. (1989 ). All electroporation experiments were carried out using a GenePulser apparatus (Bio‐Rad Laboratories, Richmond, CA, USA).

Dineen R.L., Penno C., Kelleher P., Bourin M.J., O'Connell‐Motherway M, & van Sinderen D. (2021). Molecular analysis of the replication functions of the bifidobacterial conjugative megaplasmid pMP7017. Microbial Biotechnology, 14(4), 1494-1511.

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