All procedures were carried out using the miRCat™ microRNA Cloning Kit (IDT, Iowa, USA) protocol as basis, with the modifications described in [25 (link)]. For the enrichment of the small RNA fraction, slices were cut from a 12% denaturing (7 M Urea) polyacrylamide gel, using a miSPIKE™ (IDT, Iowa, USA), as internal size marker. Small RNA fractions were purified using Performa DTR gel filtration cartridges (EdgeBio, Gaithersburg, USA). A double ligation was carried out using 3′ and 5′ linkers from miRCatTM kit (IDT, Coralville, USA) in two steps. The 3′ preadenylated linker was coupled to the small RNA fraction in a reaction in which the mix contained 1X Reaction Buffer for T4 Ligase (Thermo Fisher Scientific) without ATP, 0.1 mg BSA (Thermo Fisher Scientific), 1 U T4 RNA ligase (Thermo Fisher Scientific) and 2.5 μM 3′ linker. 6.5 μL small RNA fraction was added and incubated at 37 °C for 2 h. The 5′ linker was coupled in a mix with 1X ligation buffer (Thermo Fisher Scientific), 1 U T4 RNA ligase (Thermo Fisher Scientific) and 50 μM 5′ linker in presence of ATP. 7.5 μL 3′linked RNA was added to a final volume of 10 μL. The mixture was incubated for 2 h at 37 °C.
Ligation buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Ligation buffer is a solution used in molecular biology experiments to facilitate the joining of DNA fragments through the process of ligation. It provides the necessary chemical conditions for the DNA ligase enzyme to catalyze the formation of phosphodiester bonds between the ends of DNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
T4 rna ligase, by Thermo Fisher Scientific (1 mentions)
Reaction buffer for t4 ligase, by Thermo Fisher Scientific (1 mentions)
Ferrous sulphate, by Merck Group (1 mentions)
Hind 3, by Merck Group (1 mentions)
Ferric chloride, by Merck Group (1 mentions)
Agarose gel, by Thermo Fisher Scientific (1 mentions)
Citric acid, by Merck Group (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Glycerol, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Thermo Fisher Scientific (1 mentions)
Ssc buffer, by Merck Group (1 mentions)
Calcium chloride, by Merck Group (1 mentions)
Tris acetate buffer, by Thermo Fisher Scientific (1 mentions)
Midiprep kit, by Thermo Fisher Scientific (1 mentions)
Blue orange loading dye, by Promega (1 mentions)
Ammonium citrate tribasic, by Merck Group (1 mentions)
Bacto yeast, by Merck Group (1 mentions)
7 protocols using ligation buffer
1
microRNA Cloning Using miRCat Kit
2
Molecular Cloning Protocol: Transformation and Plasmid Purification
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Bactotryptophan (code 91079-40-2); Bacto yeast (code 8013-01-2); Sodium Chloride (code 7647-14-5); Agar (code 9002-18-0); Calcium Chloride (code 10035-04-8); Isopropanol (code 67-63-0); Ethanol (code 64-17-5); SSC Buffer (code 6135-04-3); Hind III (code 81295-22-9); BGL II (code 81295-12-7); Ammonium Citrate Tribasic (code 3458-72-8); Citric Acid (code 77-92-9); Lysine (code 56-87-1); Ferrous Sulphate (code 7720-78-7) were all from Sigma Aldrich. Glycerol (code 56-81-5); Midiprep Kit, (code K0841); Agarose Gel (code 9012-36-6); Propidium Iodide (code 25535-16-4); DNA Digestion Kit (code AM1907); Ligation Buffer (code IVGN2104) were from Thermo Fischer Scientific. Ampicillin (code 69-52-3) and Tris Acetate Buffer (code 135852-26-5) were from Fischer Scientific; Blue-Orange Loading Dye was from Promega; Miniprep Kit was from Euro genomics and Ferric Chloride (code 7705-08-0) was from Merck Millipore.
3
Circular ssDNA Synthesis via DNA/RNA Ligation
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DNA-templated DNA ligation and RNA-templated DNA ligation were performed to create the circular ssDNA. Both RNA- and DNA-templated DNA ligation were performed in a volume of 20 μL containing 2 μL of T4 DNA ligase (5 U/μL; Thermo Fisher, USA), 2 μL 10 × ligation buffer (Thermo Fisher), 1 μL PEG 4000, 1 μL padlock probe (10 pmol), 1 μL RNA or DNA template (∼10 pmol) and 13 μL DEPC water (LABTOP BIO, Shanghai, China). The ssDNA or RNA-template DNA ligation reaction was incubated at 37 °C for 30 min. In the dsDNA-template DNA ligation reaction, the dsDNA and padlock probe were pre-incubated at 95 °C for 5 min before adding T4 DNA ligase. The ligation products were analyzed by 2% (w/v) agarose gel electrophoresis with GelRed staining.
4
Molecular Diagnostics Assay Development
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T4 DNA ligase, 10× ligation buffer, bovine serum albumin (BSA), ATP, GeneRuler low range DNA ladder, SYBR Gold gel stain, Tris-HCl buffer (1 M, pH 8.0), and Tris-acetate-EDTA buffer (TAE, 50×) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Bst 3.0 polymerase, Bst buffer (isothermal amplification buffer II, 10×), MgSO 4 , Nb.BtsI nickase, and loading buffer were obtained from New England BioLabs (Ipswich, MA, USA). Fetal bovine serum (FBS) and agarose were obtained from Sigma-Aldrich (St. Louis, MO, USA). Streptavidin-coated cross-linked hydroxyethyl starch iron oxide composite particles (100 nm size MNP, product code 10-19-102) were supplied by Micromod Partikeltechnologie GmbH (Rostock, Germany). Sequences of target, PLP, and detection probes listed in Table S1 were synthesized by Integrated DNA Technologies (Coralville, IA, USA) and dissolved in 50 mM Tris-HCl (pH 8.0).
5
Genome-wide Adaptor Ligation Protocol
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Adaptors N Bam 12 (5′-GATCCTCCCTCG-3′) and N Bam 24 (5′-AGGCAACTGTGCTATCCGAGGGAG-3′) (32 (link)) were ligated to Mbo I-digested human genomic DNA. The reaction included 100 pmols of each primer, 1 μg DNA template, 3 μl ligation buffer (Fermentas) and 1,3 mM MgCl2 in a final volume of 28 μl. The sample was heated to 55°C and the temperature lowered to 4°C at the rate of 1°C/min. The sample was put on ice and 800 U of T4 DNA ligase (Fermentas) added at 4°C for a final volume of 30 μl. The ligation reaction was at 15°C for 16 h. After ligation, polymerase extension was carried out at 68°C for 10 min, with dNTP’s (0.2 mM), 4 μl 10 × polymerase buffer (Finnzymes) and 4 U Dynazyme polymerase (2 U/μl, Finnzymes) in a final volume of 40 μl.
6
Enzymatic Ligation of Biotinylated, Labeled RNA
7
Cloning of Gene from Haloalkaliphilic Bacteria
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For the sequencing using M13 forward and reverse primers, the amplified fragment was first cloned into pTZ57R/T vector and sub-cloned into pUC19 vector. In order to sub-clone into a pUC19 vector, both the plasmids from the clones and pUC19 were double-digested with EcoRI and SalI enzymes. Both the products were purified as per the manufacturer's instructions by the partition chromatography using silica gel-based plasmid DNA purification kit (Hi-media Laboratories). A ligation reaction was set up using both vector control and vector with insert in a ratio of 3:1 using T4 DNA ligase enzyme, ligation buffer (Fermentas) according to manufacturer's instructions. The competent cells of E. coli DH5α were used for the transformation of the ligated vector. The transformation by the heat shock treatment at 50 °C for 30 s was followed by cold shock on ice for 2 min. pUC-Ve 2 -BHAP-TA clones of the amplified gene from haloalkaliphilic bacteria Ve 2 -20-9 1 were obtained. The cloning was confirmed by plasmid isolation and restriction analysis of the randomly selected transformants.
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