Restriction enzyme

Manufactured by New England Biolabs

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Restriction enzymes are specialized proteins that recognize and cleave specific DNA sequences, known as restriction sites, within a DNA molecule. These enzymes are essential tools in molecular biology and genetic engineering, enabling the manipulation and analysis of DNA sequences.

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Lab products found in correlation

748 protocols using restriction enzyme

Plasmid Construction and Mutagenesis

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The sgRNA against human FBXW7 and small interfering RNA against human MAP4 and negative controls were synthesized by IGE BIOTECH (China) (Supplementary Table 1).
Wild-type ORFs for FBXW7 or CHEK1 were PCR-amplified from ESCC cell lines. The ORF of FBXW7 was inserted into pcDNA3.1 cloning vectors by restriction enzyme (NEB, USA). The sequence of the FBXW7 WD40 domain was inserted into pGEX-6P-1 cloning vectors by restriction enzyme (NEB, USA). The ORF of CHEK1 was inserted into pEGFP-C1 cloning vectors by restriction enzyme (NEB, USA). The expression plasmid for MAP4 was cloned in a previous study [19 (link)]. Various mutant plasmids were generated using a KO-Plus- Mutagenesis Kit (Toyobo, Japan) (Supplementary Table 2).

Pan Y., Liu J., Gao Y., Guo Y., Wang C., Liang Z., Wu M., Qian Y., Li Y., Shen J., Lu C, & Ma S. (2023). FBXW7 loss of function promotes esophageal squamous cell carcinoma progression via elevating MAP4 and ERK phosphorylation. Journal of Experimental & Clinical Cancer Research : CR, 42, 75.

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Molecular Cloning and Sequencing Protocol

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Antibiotics were obtained from Sigma. Targocil and targocil-II were synthesized as previously described^{16 (link), 17 (link)}. Targocil-II can also be purchased from Aurora Fine Chemicals LLC. Restriction enzymes were obtained from New England Biolabs. DNA concentrations were measured using a NanoDrop One Spectrophotometer (Thermo Scientific). Plasmid DNA was irradiated using a UV-Crosslinker (CX-2000, UVP). Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs, and PCR was performed with KOD Hotstart DNA polymerase (Novagen) on genomic DNA isolated from wildtype RN4220 S. aureus. PCR products and plasmids were purified using Qiagen kits. Primers were designed using DNASTAR-Lasergene, obtained from Integrated DNA Technologies (IDT), and are listed in Table S2. DNA sequencing was completed through Eton Bioscience and the Dana-Farber/Harvard Cancer Center DNA Sequencing Facility. For colony PCR, a colony was resuspended in 30 μL of buffer containing 40 mM NaOH and 0.2% SDS, and incubated at 98°C for 5 min followed by cooling on ice for 5 min. After dilution with 200 μL H₂O, 4 μL of this cell lysate was used for a 25 μL PCR reaction.

Lee W., Do T., Zhang G., Kahne D., Meredith T.C, & Walker S. (2018). Antibiotic combinations that enable one-step, targeted mutagenesis of chromosomal genes. ACS infectious diseases, 4(6), 1007-1018.

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Gene editing analysis using restriction enzymes

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Restriction enzymes (all from NEB) were also used to assess gene editing. Digestion reactions for all Restriction enzymes were incubated at 37 °C for 1 h in CutSmart Buffer with the exception of SspI that was incubated in NEB Buffer 2.1. Digestion products were visualised in 2% agarose gel in 1xTBE buffer.

Lataniotis L., Albrecht A., Kok F.O., Monfries C.A., Benedetti L., Lawson N.D., Hughes S.M., Steinhofel K., Mayr M, & Zampetaki A. (2017). CRISPR/Cas9 editing reveals novel mechanisms of clustered microRNA regulation and function. Scientific Reports, 7, 8585.

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Recombinant Protein Expression in E. coli

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BocLys was purchased from Bachem. K-alkyne and K-alkene were synthesized by following previously reported method (22 (link)). Primers were ordered from Sigma. Restriction enzymes, Gibson Assembly reagents, Restriction enzymes, and T4 DNA ligase were purchased from New England Biolabs. KOD hot start DNA polymerase was purchased from EMD Millipore. Standard molecular biology techniques (23 ) were used throughout. Site-directed mutagenesis was carried out using overlapping PCR. Gibson Assembly or ligation was used for cloning. Escherichia coli GeneHogs (Thermo Fisher Scientific Inc) were used for routine cloning and DNA propagation. E. coli C321.ΔA.exp (Addgene) or GeneHogs were used for libraries screening and evaluation. All solutions were prepared in deionized water further treated by Barnstead Nanopure® ultrapure water purification system (Thermo Fisher Scientific Inc.). Antibiotics were added where appropriate to the following final concentrations: ampicillin, 100 mg L⁻¹; kanamycin, 50 mg L⁻¹.

Chen Y., He X., Ma B., Liu K., Gao T., Niu W, & Guo J. (2022). Noncanonical amino acid mutagenesis in response to recoding signal-enhanced quadruplet codons. Nucleic Acids Research, 50(16), e94.

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Genetic Locus Integration Validation

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Southern blot analysis from wild type line 820 and three different ap2-g length-variable knockouts was performed to show successful integration of the selectable marker cassette at the desired genetic locus. Briefly, approximately 10 μg of Plasmodipur (EuroProxima) filtered and purified genomic DNA from lines 820 (WT), G401cl1 (complete orf knockout), G418cl6cl3 (DNA-binding domain knockout) and G529cl2 (partial orf knockout bearing the GNPm7, 8 and 9 mutations) was double-digested each with 7 μl of appropriate restriction enzyme (New England Biolabs) pairs at 37°C for 4 hours with NEB Buffer4. For comparison with the WT line (820), gDNA from WT and G401cl1, WT and G418cl6cl3 & WT and G529cl2 was double-digested with the High-Fidelity (HF) versions of NcoI & SpeI, NcoI & BamHI and EcoRI & SpeI, respectively. After transfer the membrane was hybridized (60°C overnight) with P^{32 (link)} labelled single-stranded DNA probe for a specific region from one of the homology arms used for generating the gene targeting vector. The probes were PCR-amplified and purified using the following oligonucleotides: GU1058/GU1059 for G401cl1, GU1416/GU1417 for G418cl6cl3 and GU1414/GU1415 for G529cl2. The membrane was washed thrice with decreasing concentration of SSC (3× SSC, 1× SSC, 0.5×SSC) and exposed to a maximum resolution X-ray film (BioMax MR film; Kodak) for 35 hours.

Sinha A., Hughes K.R., Modrzynska K.K., Otto T.D., Pfander C., Dickens N.J., Religa A.A., Bushell E., Graham A.L., Cameron R., Kafsack B.F., Williams A.E., Llinas M., Berriman M., Billker O, & Waters A.P. (2014). A cascade of DNA binding proteins for sexual commitment and development in Plasmodium. Nature, 507(7491), 253-257.

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Construction and Validation of E. coli CFT073 Deletion Mutants

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E. coli CFT073 deletion mutants (Table 1) were constructed using the lambda red recombinase system [55 (link)]. Primers homologous to sequence within the 5’ and 3’ ends of the gene to be targeted were designed to replace the gene with a nonpolar kanamycin-or chloramphenicol-resistant cassette amplified from template plasmid pKD4 or pKD3, respectively [55 (link)]. E. coli CFT073 mutants were confirmed by PCR amplification using primers flanking the target gene sequence and comparing gene product size to wild-type CFT073 PCR product size. To decipher negligible product sizes PCR reactions were digested with a restriction enzyme (New England Biolabs). The antibiotic resistance cassette of the fumC mutant was removed with plasmid pCP20 to unmark the strain for deletion of additional genes; fumA and/or fumB. Oligonucleotides used to construct and confirm the fumA, fumB, fumAB, fumAC, fumBC, fumABC, and sdhBfrdA mutant strains are listed in Table S1.

Himpsl S.D., Shea A.E., Zora J., Stocki J.A., Foreman D., Alteri C.J, & Mobley H.L. (2020). The oxidative fumarase FumC is a key contributor for E. coli fitness under iron-limitation and during UTI. PLoS Pathogens, 16(2), e1008382.

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Plasmid Purity Verification by Restriction Enzyme Fingerprinting

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The purity of each plasmid sample to be sequenced was confirmed by restriction enzyme fingerprinting prior to library construction. A pair of restriction enzymes was chosen to generate a unique banding pattern for each construct. Reactions were set up with 2.5 µg plasmid, 180 total units of restriction enzyme (New England Biolabs), plus 1 unit of Topoisomerase I (New England Biolabs, M0301S) to promote complete digestion. Digestions were run according to the manufacturer’s instructions. The resulting banding patterns were visualized on 0.8% E-gels (Thermofisher, G501808). A representative gel is shown in Additional file 2: Figure S3.

Brown S.D., Dreolini L., Wilson J.F., Balasundaram M, & Holt R.A. (2023). Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device. BMC Bioinformatics, 24, 116.

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Global DNA Methylation Quantification

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Global DNA methylation was determined using the LUMA, which is based on the ability of two isoschizomers (MspI and HpaII) to digest sequences differentially depending on the methylation status of the CpG site contained within the sequence as described in Karimi et al. (2006a) (link). In brief, 300 ng of genomic DNA were cleaved at 37 °C for 4 h and 80 °C for 20 min with HpaII/EcoRI or MspI/EcoRI in two separate 20 μl reactions containing 2 μl of Tango buffer (Thermo Scientific, Waltham, MA) and five units of each restriction enzyme (NEB, Ipswich, MA). Fifteen microliters of annealing buffer (20 mM TRIS-acetate, 2 mM Mg-acetate pH 7.6) were mixed with the digested samples and placed in a PyroMark Q96 MD system. The dispensation order used for sequencing was: GTGTCACATGTGTG. For calculations, the peak heights of dispensations 9 and 10 were used. Samples with peaks lower than 3, the cut-off value for DNA quality, were discarded. Percentage of DNA methylation was expressed as [1 – (HpaII/EcoRIΣG/ΣT)/(MspI/EcoRIΣG/ΣT)] × 100. The samples were analyzed in technical duplicates and each plate included a positive, negative and water control. Lambda DNA (NEB) was used as a negative control and methylated Lambda DNA using M.SssI methyltransferase was used as a positive control. All samples used in the final analysis had an intra-assay coefficient of variation of ≤5%.

Brown T.A., Lee J.W., Holian A., Porter V., Fredriksen H., Kim M, & Cho Y.H. (2015). Alterations in DNA methylation corresponding with lung inflammation and as a biomarker for disease development after MWCNT exposure. Nanotoxicology, 10(4), 453-461.

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Genotyping of TLR Polymorphisms by PCR-RFLP

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SNPs of each DNA sample were determined by PCR-based restriction fragment length polymorphism (RFLP). PCR was carried out in a 25–30 ul mixture containing 1 ug of genomic DNA, Go Taq Green Master Mix (400uM of dATP, dCTP, dTTP, dGTP, Taq polymerase, 3mM MgCl₂; Promega Corp., Madison WI) and 1ul each of 0.1uM primer (Sigma Chemical Co., St. Louis MO), detailed on Table 1. PCR was performed in a thermocycler with denaturation and annealing steps, varied for each TLR polymorphism following published methods [21 (link)–23 (link)]. 3ul of PCR products were digested overnight and mixed with 7ul of restriction enzyme (New England Biolabs Inc., Ipswich, MA) for each (Table 1). 10 ul of PCR product was loaded on agarose gels, varied for each TLR polymorphism according to published methods [24 (link)–26 (link)] including 1ul loading buffer (EMD Chemicals, San Diego, CA) (Fig 1). Representative patient samples are shown in each lane of each panel of Fig 1. DNA quantity was insufficient for analysis for some samples of each group. For clarity, each SNP designation is also provided in Table 1 as given in the public single-nucleotide polymorphism database (dbSNP).

Berenson C.S., Kruzel R.L., Wrona C.T., Mammen M.J, & Sethi S. (2015). Impaired Innate COPD Alveolar Macrophage Responses and Toll-Like Receptor-9 Polymorphisms. PLoS ONE, 10(9), e0134209.

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PCR and Restriction Enzyme Analysis

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PCR was carried out on DNA extracted from eight plants per population using PuReTaq Ready-To-Go PCR beads (Amersham Biosciences, Bucks, UK) in a total volume of 25 µL containing 0.8 µM of each primer and about 50 ng of genomic DNA. The reactions were performed on a Master Cycle Gradient Thermocycler Model 96 (Eppendorf, UK). A hot start was applied with a denaturation at 95 °C for 4 min followed by 30 cycles of 30 s at 95 °C, 30 s at 60 °C and 1 min at 72 °C. A final extension step for 10 min at 72 °C was also included. Three microlitres of neat PCR product was digested with 7.5 units of each restriction enzyme (New England Biolabs, Hertfordshire, UK) in a total volume of 20 µl according to the manufacturer’s recommendations and analysed on 4% MetaPhor^TM agarose gel (Lonza, Walkersville, MD, USA) containing 0.5 µg mL⁻¹ ethidium bromide run for 1 h at 80 V with 1× TBE buffer.

Kaundun S.S., Marchegiani E., Hutchings S.J, & Baker K. (2019). Derived Polymorphic Amplified Cleaved Sequence (dPACS): A Novel PCR-RFLP Procedure for Detecting Known Single Nucleotide and Deletion–Insertion Polymorphisms. International Journal of Molecular Sciences, 20(13), 3193.

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