Peripheral venous blood was drawn from each participant. Genomic DNA was extracted from whole blood using the Maxwell 16 DNA Purification Kit (Promega Corporation, Madison, WI). The choice of SNP was informed by the results of a prior analysis of WNK1. All the genotyping experiments were done by the Shanghai Generay Biotech Co., Ltd. (http://www.generay.com.cn/ ) using ligase detection reactions (LDR). The target DNA sequences were amplified using a multiplex PCR method. After the completion of the amplification, 1 μl of proteinase K (20 mg/ml) was added, then heated at 70°C for 10 min and quenched at 94°C for 15 min. The ligation reaction for each subject was carried out in a final volume of 10 μl containing 2 μl of multi-PCR product, 1 μl of probe, 0.125 μl of 40 U/μl Taq DNA ligase (NEB, USA), 1 μl of 10× Taq DNA ligase buffer, and 6 μl H2O. The LDR was performed using 25 cycles of 94°C for 30 sec and 55°C for 4 min. The fluorescent products of LDR were differentiated by use of an ABI sequencer 377. Additionally, about 5% of the samples were randomly selected and retested by direct DNA sequencing on a 3730xl DNA analyzer (Applied Biosystems) and the results were 100% concordant.
Taq dna ligase
Manufactured by New England Biolabs
Sourced in United States, China
Taq DNA ligase is a thermostable enzyme used for the covalent joining of double-stranded DNA fragments. It catalyzes the formation of a phosphodiester bond between adjacent 3'-hydroxyl and 5'-phosphate termini in duplex DNA or RNA.
Automatically generated - may contain errors
Lab products found in correlation
T5 exonuclease, by New England Biolabs (20 mentions)
Phusion dna polymerase, by New England Biolabs (11 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (8 mentions)
Taq dna polymerase, by New England Biolabs (7 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (5 mentions)
T4 dna ligase, by New England Biolabs (5 mentions)
Nt bspqi, by New England Biolabs (3 mentions)
T5 exonuclease, by Illumina (3 mentions)
Nebuffer 3, by New England Biolabs (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Restriction enzyme, by New England Biolabs (2 mentions)
Thermopol buffer, by New England Biolabs (2 mentions)
Bst dna polymerase, by New England Biolabs (2 mentions)
Bovine serum albumin (bsa), by New England Biolabs (2 mentions)
Bst 2.0 dna polymerase, by New England Biolabs (2 mentions)
Taq dna ligase reaction buffer, by New England Biolabs (2 mentions)
T4 polynucleotide kinase, by New England Biolabs (2 mentions)
Dpni, by New England Biolabs (2 mentions)
T7 dna ligase, by New England Biolabs (2 mentions)
Q5 dna polymerase, by New England Biolabs (2 mentions)
63 protocols using taq dna ligase
1
Genotyping of WNK1 Variants
2
Recombinase-Mediated Variant Generation
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Typically donor primer: ssDNA ratios around 1000:1 work well; lower ratios will also work, but as the primer: template ratio decreases there is an increased likelihood of skipping a primer and obtaining the template sequence instead of the desired variants. The amount of ssDNA template used here depends in part on the previous UDG test. Typically, for the recombinase study, we used ssDNA template at a concentration of ∼2.5 ng/µl or ∼10 fmol/µl. In this step, annealing occurs at room temperature, which favors random annealing of the primer mixtures to the template. Typically 10 µl annealing reactions are performed in PCR tubes. Each reaction contains 1 µl 10× Taq ligase buffer, 10 fmol ssDNA, 10 pmol of 5′ PCR forward primer, 10 pmol of donor primer mixture, and water to 10 µl total. Incubate at room temperature for 30′. For the extension and ligation reaction, in a PCR tube, place 1 µl of the annealed sample, 1 µl 10× Taq DNA ligase buffer, dNTP mixture for a final concentration of 100 µM, 2.5 units Taq DNA ligase (New England Biolabs, M0208S), 0.75 units Phusion-U-Hotstart polymerase, water to 10 µl. Incubate at 55°C for 30′.
3
Ligase-Mediated Rolling Circle Amplification
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The ligation was carried out in a 10 μL mixture containing: 1 × Taq DNA ligase buffer (20 mM Tris–HCl, 25 mM KAc, 10 mM Mg(Ac)2, 10 mM DTT, 1 mM NAD, 0.1% Triton X-100), 10 pM linear padlock probe, 12 U of Taq DNA ligase (NEB, United States) and 1 μL of DNA template (10 ng). The ligation mixture was incubated at 65°C for 1 h.
After ligation, 1 μL of ligation product was added into an HRCA reaction mixture containing 1 × ThermoPol buffer (20 mM Tris–HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100), 0.4 mM dNTP mix, 0.5 μM of each HRCA primers and 1.6 U Bst DNA polymerase (NEB, United States) with a total 10 μL volume. The reaction was performed in a 0.2 mL tube in a water bath incubated at 62°C for 60 min.
The results were judged by the appearance of color after adding 1 μl of 1000 × SYBR Green I dye to the system after the reaction or 2% agarose gel electrophoresis of the HRCA product.
After ligation, 1 μL of ligation product was added into an HRCA reaction mixture containing 1 × ThermoPol buffer (20 mM Tris–HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100), 0.4 mM dNTP mix, 0.5 μM of each HRCA primers and 1.6 U Bst DNA polymerase (NEB, United States) with a total 10 μL volume. The reaction was performed in a 0.2 mL tube in a water bath incubated at 62°C for 60 min.
The results were judged by the appearance of color after adding 1 μl of 1000 × SYBR Green I dye to the system after the reaction or 2% agarose gel electrophoresis of the HRCA product.
4
Magnetic Bead-based DNA Ligation Assay
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After hybridization, magnetic beads were washed twice using washing buffer 1 (2× SSC, 0.5% SDS) and washing buffer 2 (2× SSC), respectively, to remove excess and mishybridized LSPs. Then, 25 µL master mix containing 0.4 units Phusion high-fidelity DNA polymerase (NEB), 40 units Taq DNA ligase (NEB), 1 mM NAD (NEB), 0.1 mM dNTPs, 1× Phusion HF Buffer was added to the beads. The reaction was incubated for 20 min at 45°C to allow upstream LSP1s to extend and ligate to downstream LSP2s. After extension and ligation, the beads were washed once with elution buffer (10 mM Tris-Cl, pH 8.5), then resuspended in 35 µL elution buffer and heated for 1 min at 95°C to release the ligated products.
5
Molecular Biology Protocol Reagents
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All chemicals and reagents were purchased from Thermo Scientific (Pittsburgh, PA) or Sigma-Aldrich (Saint Louis, MO) unless otherwise specified. Taq DNA ligase, Phusion High-Fidelity DNA polymerase, and T5 exonuclease were obtained from New England Biolabs (Ipswich, MA). Oligonucleotides were purchased from IDT (San Diego, CA). KOD DNA polymerase was purchased from EMD Chemicals (San Diego, CA).
6
DNA Amplification and Modification Reagents
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All chemicals and reagents were purchased from Sigma-Aldrich (Saint Louis, MO) or Thermo Scientific (Pittsburgh, PA) unless specified otherwise. KOD DNA polymerase was purchased from EMD Chemicals (San Diego, CA). Taq DNA ligase, Phusion High-Fidelity DNA polymerase, and T5 exonuclease were obtained from New England Biolabs (Ipswich, MA). Oligonucleotides were purchased from IDT (San Diego, CA).
7
CRISPR-Cas9 Plasmid Assembly Protocol
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The cleaved backbone plasmid were prepared by in vitro cleavage using CRISPR-Cas9 and appropriate PCR product were amplified using PCR templates; pSpCas9(BB)-2A-Puro (PX459) was a gift from Feng Zhang (Addgene plasmid # 48139; http://n2t.net/addgene:48139 ; RRID:Addgene_48139), MSP469 was a gift from Keith Joung (Addgene plasmid # 65771; http://n2t.net/addgene:65771 ; RRID:Addgene_65771) and MSP1101 was a gift from Keith Joung (Addgene plasmid # 65773; http://n2t.net/addgene:65773 ; RRID:Addgene_65773). The backbone plasmid and insert PCR product mixed in a volume of 10 μl containing 2U T5 exonuclease (New England Biolabs, catalog number: M0363S), 12.5U phusion DNA polymerase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: F530L), 2kU Taq DNA ligase (New England Biolabs, catalog number: M0208S), 0.2 M Tris-HCl (pH 7.5), 0.2 M MgCl2, 2 mM dNTP, 0.2 M dithiothreitol, PEG 8000, and 1 mM NAD and incubated at 50 °C for 1 hour. The products were then transformed into 100 μl of DH5α competent cells. Single transformed colonies were inoculated into LB medium containing antibiotics. DNA products were isolated from cells using a DNA prep kit (MGmed).
8
Comparative Analysis of DNA Assembly Methods
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Phusion DNA polymerase (referred as ‘Phusion’ in text, Thermo Fisher, Beijing) was used to amply DNA fragments. T5 exonuclease (New England Biolab, Beijing) was used in Gibson, Hot Fusion, and TEDA. Premix PrimeSTAR HS polymerase (referred as ‘PrimeSTAR’ in text, Takara, Japan) and TransStart FastPfu Fly DNA Polymerase (referred as ‘FastPfu’ in text, Transgen, Beijing) were used in testing the Gibson and Hot Fusion methods. Taq DNA ligase (New England Biolab) was used to prepare the self-produced Gibson reaction mixture. 2X HotStart Taq plus Master Mix (Novoprotein, Beijing) was used for colony PCR test. The 5- and 1-kb DNA markers of Transgen (Beijing) were used to estimate DNA fragments. DNA and PCR product purification kits (Omega, US) were used to purify DNA fragment. All other chemicals were purchased from Sangon Biotech except PEG8000 that was purchased from Sigma-Aldrich (Shanghai). All restriction enzymes were purchased from Thermo Fisher. All oligos were synthesized by Beijing Genomics Institute.
9
Bacterial Expression Vectors for Protein Modification
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For expression in bacterial cells pRSFDuet-1 (Merck/Sigma-Aldrich, Novagen) and a vector derived from pGEX-4T-1 (GE Healthcare) were used and modified as described above (Supplementary Data 4 ). Mutations were introduced by site-directed mutagenesis according to QuickChange protocol (Supplementary Table 8 ). For cloning, Phusion-DNA-polymerase, Taq-DNA-ligase, T5 exonuclease, and restriction enzymes were used (New England Biolabs). Anti-AcK-, anti-His6-, anti-FLAG-primary antibodies and suitable HRP-coupled secondary antibodies were purchased from Abcam, Cell Signaling Technologies and Invitrogen (rabbit anti-AcK-AB: abcam, ab21623/dilution: 1:1000 in 3-5% (w/v) milk; mouse anti-His6-AB: abcam, ab18184/dilution: 1:000 in 3-5% (w/v) milk; rabbit anti-FLAG-AB: Cell Signaling Technology, CST14793/dilution: 1:1000 in 3–5% (w/v) milk; mouse anti-FLAG-AB: Invitrogen, FG4R/dilution: 1:2000 in 3% (w/v) milk; goat anti-rabbit-HRP-AB: abcam, ab6721/dilution: 1:10000 in 3-5% milk; rabbit anti-mouse-HRP-AB: abcam, ab6728/dilution: 1:10000 in 1-5% (w/v) milk).
10
Phage Display Peptide Screening
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