Hindiii and xhoi

Manufactured by New England Biolabs

Sourced in China

HindIII and XhoI are Type II restriction enzymes commonly used in molecular biology. HindIII recognizes and cleaves the DNA sequence 5'-AAGCTT-3', while XhoI recognizes and cleaves the DNA sequence 5'-CTCGAG-3'. These enzymes are used to generate DNA fragments for cloning, genetic engineering, and other molecular biology applications.

Automatically generated - may contain errors

Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Double luciferase reporter kit, by Promega (1 mentions) Quick ligation kit, by New England Biolabs (1 mentions) Q5 hot start high fidelity 2x master mix, by New England Biolabs (1 mentions) Prime a gene kit, by Promega (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Qiaquick pcr purification kit, by Qiagen (1 mentions) Qiaprep spin miniprep kit, by Qiagen (1 mentions) Xhoi, by Qiagen (1 mentions) Dual luciferase reporter assay system, by Promega (1 mentions) Xhoi, by GenScript (1 mentions) Pet 28b vector, by GenScript (1 mentions)

Spelling variants of this product

XhoI (381 citations) HindIII (328 citations) XhoI/HindIII (2 citations) XhoI and HindIII-HF (2 citations) HindIII and XhoI restriction enzymes (2 citations) Restriction endonucleases XhoI and HindIII (2 citations)

5 protocols using hindiii and xhoi

Regulation of Mtf1 Promoter by miRNA-22

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The identified region of Mtf1 promoter from a mouse genomic DNA was amplified using primer pairs (F, 5'-ACGCTCGAGGGTGCGCCAGAGTCACTTAT-3' and R, 5'-AATAAGCTTTCTCAACCTGGT TCTGCACT-3'), and cloned into pGL6 plasmid (Beyotime, Shanghai, China) via XhoI and HindIII (NEB, USA) digestion. Empty pGL6 vector was used as control plasmid.
HEK293T cells were incubated in DMEM (Gibco BRL,Grand Island, NY, USA) containing 10% FBS (Gibco BRL) and 1 × 10⁵ cells per well were seeded into 24-well plates before transfection. The reporter plasmids pGL6 or pGL6-Mtf1 were co-transfected with miRNA-22 mimics or miRNA-22 inhibitor into 293 cells using Lipofectamine 2000 (11668-027, Invitrogen). After 48 h of incubation, the cells were collected to detect luciferase activity assays using the Double luciferase reporter kit (E1910, Promega, Madison, WI, USA) in accordance with the manufacturer's instruction. The ratio of firefly luciferase activity to Renilla luciferase activity in each group (Renilla luciferase activity as an internal reference) was used as the reporter gene activity.

Hao L.Y., Zhang M., Tao Y., Xu H., Liu Q., Yang K., Wei R., Zhou H., Jin T., Liu X.D., Xue Z., Shen W., Cao J.L, & Pan Z. (2022). miRNA-22 Upregulates Mtf1 in Dorsal Horn Neurons and Is Essential for Inflammatory Pain. Oxidative Medicine and Cellular Longevity, 2022, 8622388.

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Dual-expression Plasmid Construction

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To make a dual-expression plasmid, our in-house pET-dual plasmid containing the gene of interest was amplified by PCR to add XhoI and HindIII cutting sites with Q5^® Hot Start High-Fidelity 2X Master Mix (NEB). The amplified gene fragment and the same vector with the other gene of interest were cut by XhoI and HindIII (NEB) and gel purified. The amplified gene fragment was then ligated into the vector by Quick Ligation Kit (NEB).

Evans D, & Blumenthal T. (2000). trans Splicing of Polycistronic Caenorhabditis elegans Pre-mRNAs: Analysis of the SL2 RNA. Molecular and Cellular Biology, 20(18), 6659-6667.

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Northern Blot Analysis of RNA

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Total RNA was isolated from transfected cells using Tri reagent (MRC) according to the manufacturer's protocol. The RNA was separated on a denaturing 1% agarose gel in MOPS buffer. The gel was stained with ethidium bromide to visualize total RNA (mostly comprising of rRNA) as a loading control. The RNA was transferred overnight to a nitrocellulose membrane. Subsequently, the membrane was UV crosslinked and the RNA hybridized to a ³²P-labeled probe overnight in Church buffer 65°C. The membrane was imaged using a Typhoon phosphoimager system. The probe was generated first restricting the plasmid using HindIII and XhoI (NEB), gel purifying the restricted fragment and using Prime a gene kit (Promega) in the presence of α³²P-dATP.

Akef A., Lee E.S, & Palazzo A.F. (2015). Splicing promotes the nuclear export of β-globin mRNA by overcoming nuclear retention elements. RNA, 21(11), 1908-1920.

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Cloning and Luciferase Assay of DR4 Construct

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Vector pGL4.10 [luc2] (Promega) was linearized by digesting with HindIII and XhoI (New England Biolabs), and was then purified using the QIA quick PCR purification kit (Qiagen 28104) Two ssDNA oligos (DR4 sequence with overhangs) were annealed to form a dsDNA oligo with specific HindIII and XhoI overhangs. The linearized vector and dsDNA oligo were ligated together using T4 DNA ligase (NEB) according to manufacturer protocol. The product was then transformed into competent cells and plated onto ampicillin agar plates. Single colonies were picked the following day and grown in LB overnight. The cells were then harvested and mini prepped with Qiaprep spin Miniprep Kit (Qiagen 27104). Purified plasmid DNA was sent for sequencing to confirm successful and correct ligation. A Dual-Luciferase Reporter Assay System (Promega) was used to determine DR4 luciferase activity (firefly) and each individual well was normalized via Renilla luciferase internal control.

Alves C.R., Neves W.D., de Almeida N.R., Eichelberger E.J., Jannig P.R., Voltarelli V.A., Tobias G.C., Bechara L.R., de Paula Faria D., Alves M.J., Hagen L., Sharma A., Slupphaug G., Moreira J.B., Wisloff U., Hirshman M.F., Negrão C.E., de Castro Jr G., Chammas R., Swoboda K.J., Ruas J.L., Goodyear L.J, & Brum P.C. (2020). Exercise training reverses cancer-induced oxidative stress and decrease in muscle COPS2/TRIP15/ALIEN. Molecular Metabolism, 39, 101012.

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Cloning and Expression of TTR Protein

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The construct corresponding
to the TTR amino acid sequence plasmid pcDNA3.1+/C-(K) DYK with Accession
No. NM_000371 was purchased from GenScript, USA. The TTR gene was
cloned as per the protocol described by Ali et al.^{48 (link)} The plasmid pcDNA3.1+/C-(K) was amplified using PCR with
this primer set: the primers 5′-ATATATAAGCTTATGGCTTCTCATCGTCTG-3′
tagged with a 5′-HindIII cleavage site and
5′-ATAT ATCT CGA GT CATTCCT TGGGATTGG-3′ tagged with
a 5′ XhoI cleavage site. A construct corresponding
to the mature protein without the predicted signal sequence was amplified.
The PCR-amplified product was double-digested with HindIII and XhoI restriction enzymes. The pET28b vector
(GenScript) was also double-digested with HindIII
and XhoI restriction enzymes. The digested product
(insert and backbone) was ligated using the T4 DNA ligase. The ligated
vector was used to transform the Escherichia coli DH5α strain, which was plated onto Luria–Bertani (LB)
agar plates containing 50 μg/mL of kanamycin (Km). The subsequent
transformants were collected for plasmid preparation using Gene Jet
Minipreps (Thermo Scientific). The plasmids were digested with HindIII and XhoI (New England Biolabs)
to confirm the positive clones. The constructs were sequenced using
Eurofins.

Ali A., Zhaliazka K., Dou T., Holman A.P, & Kurouski D. (2023). Role of Saturation and Length of Fatty Acids of Phosphatidylserine in the Aggregation of Transthyretin. ACS Chemical Neuroscience, 14(18), 3499-3506.

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