t4 ligase  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Ambion T4 RNA Ligase
    Description:
    Ambion T4 RNA Ligase catalyzes the formation of a phosphodiester linkage between a 5'-phosphoryl-terminated nucleic acid donor and a 3'-hydroxyl-terminated nucleic acid acceptor. Substrates include RNA, DNA, and oligonucleotides. One tube containing 2,500 U (5 U/ µL) is provided along with 10X Reaction Buffer. The enzyme can be used for tagging the 5' ends of mRNA with oligonucleotides for mapping studies (RACE), 3'-end labeling RNA molecules, and circularizing RNA and DNA molecules.Unit Definition:One unit catalyzes the formation of 1 nmol of [5'-32P]-rA12-18 into a phosphatase-resistant form in 30 min at 37°C.
    Catalog Number:
    AM2141
    Price:
    None
    Applications:
    DNA & RNA Purification & Analysis|Nucleic Acid Labeling & Oligo Synthesis|RNA Labeling
    Size:
    2 500 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, DNA⁄RNA Modifying Enzymes
    Score:
    85
    Buy from Supplier
    Name:
    T4 DNA Ligase (1 U/µL)
    Description:
    T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate termini. The unique T4 DNA Ligase buffer optimizes ligation, which can be performed in 5 minutes. Single-stranded nucleic acids are not substrates for this enzyme.ApplicationsCloning (blunt-end or cohesive-end ligation) and adding linkers or adapters to blunt-ended DNASourcePurified from E. coli lambda lysogen NM989Performance and quality testingEndodeoxyribonuclease, 3´ and 5´ exodeoxyribonuclease assays; ligation efficiency testedUnit definitionOne unit catalyzes the exchange of 1 nmol 32P-labeled pyrophosphate into ATP in 20 min at 37°C. One unit is equal to approximately 300 cohesive-end ligation units.Unit reaction conditions66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 10 mM DTT, 66 µM ATP, 3.3 µM 32P-labeled pyrophosphate, and enzyme in 0.1 mL for 20 min at 37°C.
    Catalog Number:
    15224017
    Price:
    None
    Applications:
    ChIP-on-Chip|Cloning|RNAi, Epigenetics & Non-Coding RNA Research|Chromatin Biology|Restriction Enzyme Cloning
    Size:
    100 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, DNA⁄RNA Modifying Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher t4 ligase
    The schematic strategy for miRNA detection. Notes:  A DNAzyme-based probe contains three domains: a miRNA-binding domain (the bubble structure), a DNAzyme domain, and a substrate domain. The binding of miRNA to DNAzyme-based probe initiates T4 ligase reaction. ( A ) Secondary structure of the incomplete miRNA-specific DNAzyme. ( B ) The intact miRNA-specific DNAzyme cleaved into two fragments in the presence of Cu 2+ . ( C ) The cleaved product is then applied to formation of self-assembled DNA concatemers, eventually resulting amplified fluorescent signals in the presence of SYBR Green I. Abbreviations:  miRNA, microRNA; SA-MBs, streptavidin-coated microsphere beads.
    T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate termini. The unique T4 DNA Ligase buffer optimizes ligation, which can be performed in 5 minutes. Single-stranded nucleic acids are not substrates for this enzyme.ApplicationsCloning (blunt-end or cohesive-end ligation) and adding linkers or adapters to blunt-ended DNASourcePurified from E. coli lambda lysogen NM989Performance and quality testingEndodeoxyribonuclease, 3´ and 5´ exodeoxyribonuclease assays; ligation efficiency testedUnit definitionOne unit catalyzes the exchange of 1 nmol 32P-labeled pyrophosphate into ATP in 20 min at 37°C. One unit is equal to approximately 300 cohesive-end ligation units.Unit reaction conditions66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 10 mM DTT, 66 µM ATP, 3.3 µM 32P-labeled pyrophosphate, and enzyme in 0.1 mL for 20 min at 37°C.
    https://www.bioz.com/result/t4 ligase/product/Thermo Fisher
    Average 99 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    t4 ligase - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) Product Images from "DNAzyme-based probe for circulating microRNA detection in peripheral blood"

    Article Title: DNAzyme-based probe for circulating microRNA detection in peripheral blood

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S89560

    The schematic strategy for miRNA detection. Notes:  A DNAzyme-based probe contains three domains: a miRNA-binding domain (the bubble structure), a DNAzyme domain, and a substrate domain. The binding of miRNA to DNAzyme-based probe initiates T4 ligase reaction. ( A ) Secondary structure of the incomplete miRNA-specific DNAzyme. ( B ) The intact miRNA-specific DNAzyme cleaved into two fragments in the presence of Cu 2+ . ( C ) The cleaved product is then applied to formation of self-assembled DNA concatemers, eventually resulting amplified fluorescent signals in the presence of SYBR Green I. Abbreviations:  miRNA, microRNA; SA-MBs, streptavidin-coated microsphere beads.
    Figure Legend Snippet: The schematic strategy for miRNA detection. Notes: A DNAzyme-based probe contains three domains: a miRNA-binding domain (the bubble structure), a DNAzyme domain, and a substrate domain. The binding of miRNA to DNAzyme-based probe initiates T4 ligase reaction. ( A ) Secondary structure of the incomplete miRNA-specific DNAzyme. ( B ) The intact miRNA-specific DNAzyme cleaved into two fragments in the presence of Cu 2+ . ( C ) The cleaved product is then applied to formation of self-assembled DNA concatemers, eventually resulting amplified fluorescent signals in the presence of SYBR Green I. Abbreviations: miRNA, microRNA; SA-MBs, streptavidin-coated microsphere beads.

    Techniques Used: Binding Assay, Amplification, SYBR Green Assay

    2) Product Images from "Chromosome conformation capture of transcriptional interactions between cytochrome c oxidase genes and genes of glutamatergic synaptic transmission in neurons"

    Article Title: Chromosome conformation capture of transcriptional interactions between cytochrome c oxidase genes and genes of glutamatergic synaptic transmission in neurons

    Journal:

    doi:

    Positive and negative controls for chromosome conformation capture. All possible restriction fragments after  Bgl II  digestions were present in equimolar amounts. A. Two regions of calreticulin gene,  CalR2.8  and  CalR4.4 , were cross-linked and represented a positive control. On the other hand,  COX4i1  and  CalR  were two functionally unrelated genes, and they did not interact in our 3C reactions. B.  CalR2.8  and  CalR4.4 , as well as  COX4i1  or  COX8a  and  Grin1 ,  Gria2 , or  Nos1 , respectively, did not interact in the absence of a cross-linking agent (formaldehyde) or T4 ligase. Experiments were performed in triplicates.
    Figure Legend Snippet: Positive and negative controls for chromosome conformation capture. All possible restriction fragments after Bgl II digestions were present in equimolar amounts. A. Two regions of calreticulin gene, CalR2.8 and CalR4.4 , were cross-linked and represented a positive control. On the other hand, COX4i1 and CalR were two functionally unrelated genes, and they did not interact in our 3C reactions. B. CalR2.8 and CalR4.4 , as well as COX4i1 or COX8a and Grin1 , Gria2 , or Nos1 , respectively, did not interact in the absence of a cross-linking agent (formaldehyde) or T4 ligase. Experiments were performed in triplicates.

    Techniques Used: Positive Control

    3) Product Images from "A Rapid Cloning Method Employing Orthogonal End Protection"

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037617

    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected  13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1  to ( 13 FNIII) 8  repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8  construct.
    Figure Legend Snippet: Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Techniques Used: Incubation, Plasmid Preparation, Clone Assay, Expressing, Construct

    4) Product Images from "Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs"

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076117

    Summary of the Golden GATEway cloning kit. A) Outline of the entire cloning procedure. Eight different entry vectors (pGG-EVx) contain different inserts (colored bars). These inserts are assembled in a predefined order using a Golden Gate reaction into Gateway TM  entry vectors at any position (Threeway Gateway TM  cloning). These can then be assembled to establish a final expression vector in an LR reaction. Compatible overhangs are indicated on each Golden Gate entry vector. B) The principle of Golden Gate cloning is illustrated in the top scheme; the principle of Gateway cloning is illustrated in the bottom scheme. Golden Gate cloning utilizes type II restriction endonucleases to generate compatible overhangs that can be ligated with T4 ligase. The ligation of the two compatible inserts from the entry vectors one and two is illustrated. Gateway cloning relies on recombination of specific att sites using a commercially available enzyme mix (LR Clonase II, Life Technologies).
    Figure Legend Snippet: Summary of the Golden GATEway cloning kit. A) Outline of the entire cloning procedure. Eight different entry vectors (pGG-EVx) contain different inserts (colored bars). These inserts are assembled in a predefined order using a Golden Gate reaction into Gateway TM entry vectors at any position (Threeway Gateway TM cloning). These can then be assembled to establish a final expression vector in an LR reaction. Compatible overhangs are indicated on each Golden Gate entry vector. B) The principle of Golden Gate cloning is illustrated in the top scheme; the principle of Gateway cloning is illustrated in the bottom scheme. Golden Gate cloning utilizes type II restriction endonucleases to generate compatible overhangs that can be ligated with T4 ligase. The ligation of the two compatible inserts from the entry vectors one and two is illustrated. Gateway cloning relies on recombination of specific att sites using a commercially available enzyme mix (LR Clonase II, Life Technologies).

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Ligation

    5) Product Images from "GA-Binding Protein and p300 Are Essential Components of a Retinoic Acid-Induced Enhanceosome in Myeloid Cells"

    Article Title: GA-Binding Protein and p300 Are Essential Components of a Retinoic Acid-Induced Enhanceosome in Myeloid Cells

    Journal:

    doi: 10.1128/MCB.26.8.3060-3070.2006

    Chromosome conformation capture confirms the RA-induced CD18 enhanceosome. (A) Untreated or RA-treated U937 cells were cross-linked with 2% formaldehyde, and isolated nuclei were digested with Nsp1 overnight, followed by ligation with T4 ligase. The cross-links
    Figure Legend Snippet: Chromosome conformation capture confirms the RA-induced CD18 enhanceosome. (A) Untreated or RA-treated U937 cells were cross-linked with 2% formaldehyde, and isolated nuclei were digested with Nsp1 overnight, followed by ligation with T4 ligase. The cross-links

    Techniques Used: Isolation, Ligation

    Related Articles

    Clone Assay:

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs
    Article Snippet: We adapted the oligo annealing and cloning procedure from Life Technologies. .. The 10nM annealed double-stranded oligo dilution was used as an insert in a standard ligation reaction with T4 ligase (5U, Thermo, Fisher)

    Article Title: Splice variants of the endonucleases XPF and XPG contain residual DNA repair capabilities and could be a valuable tool for personalized medicine
    Article Snippet: Paragraph title: Cloning ... Ligation was performed in a 3:1 (fragment : vector) ratio using a T4 Ligase (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: B Cell Receptor Affinity for Insulin Dictates Autoantigen Acquisition and B Cell Functionality in Autoimmune Diabetes
    Article Snippet: For directional cloning, a 5′ primer was designed with a BglII site before the Kozak sequence and 3′ with SalI after the stop codon: Forward 5′-AATAGATCTGCCACCATGGGATGGTCATGTATCATCC-3′, Reverse 5′-TATGTCGACCTAACACTCTCCCCTGTTGAAGCTC-3′. .. The digested DNA was gel purified and ligated in multiple ratios of vector:insert with T4 ligase (Life Technologies, Carlsbad, CA, USA) overnight.

    Centrifugation:

    Article Title: GA-Binding Protein and p300 Are Essential Components of a Retinoic Acid-Induced Enhanceosome in Myeloid Cells
    Article Snippet: Following centrifugation, the cell pellet was resuspended in 50 ml lysis buffer (10 mM Tris HCl, pH 8.0, 10 mM NaCl, 0.2% NP-40 with protease inhibitors) and incubated on ice for 90 min with mixing. .. Ligation was then performed at 16°C using 30 Weiss units of T4 ligase (Invitrogen) for 4 h. Samples were then treated with proteinase K (final concentration, 100 μg/ml) and incubated overnight at 65°C to reverse cross-links.

    Amplification:

    Article Title: Splice variants of the endonucleases XPF and XPG contain residual DNA repair capabilities and could be a valuable tool for personalized medicine
    Article Snippet: Amplification fragments as well as the target vector were digested with appropriate restriction enzymes (listed in Table ) according to manufacturer’s protocol (NEB, Ipswich, MA, USA). .. Ligation was performed in a 3:1 (fragment : vector) ratio using a T4 Ligase (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: FTO Knockout Causes Chromosome Instability and G2/M Arrest in Mouse GC-1 Cells
    Article Snippet: Plasmid was ligated with annealed sgRNAs via T4 ligase (Thermo Fisher Scientific). .. For the FTO rescue experiment, total RNA was extracted from GC-1 cells using RNAiso plus Reagent (Takara Clontech). cDNA was synthesized by the first strand cDNA synthesis kit (Takara Clontech) following the manufacturer’s instructions.

    Article Title: B Cell Receptor Affinity for Insulin Dictates Autoantigen Acquisition and B Cell Functionality in Autoimmune Diabetes
    Article Snippet: Briefly, primers were designed for PCR amplification of template from the above light-chain expression vectors. .. The digested DNA was gel purified and ligated in multiple ratios of vector:insert with T4 ligase (Life Technologies, Carlsbad, CA, USA) overnight.

    Article Title: Model‐driven design of a minimal medium for Akkermansia muciniphila confirms mucus adaptation
    Article Snippet: The purified amplicon and vector pCDF‐1b (Novagen, Merck Milipore, Darmstadt, Germany) were digested using enzymes XmaIJ (Thermo Fisher Scientific) and KpnI HF (New England Biolabs) in Tango buffer (Thermo Fisher Scientific) for 1.5 h at 37°C. .. The DNA was purified again and ligated with 3:1 ratio (insert:vector) using T4 ligase (manufacturers protocol, Thermo Fisher Scientific).

    Molecular Cloning:

    Article Title: StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins
    Article Snippet: Paragraph title: DNA plasmids and molecular cloning ... Plasmids were constructed using standard molecular biology techniques: restriction enzyme digest (Fermentas), PCR and overlap extension PCR with PrimeSTAR polymerase (Clontech), and assembly using either In-Fusion enzyme (Clontech) or T4 ligase (Thermo Fisher).

    Construct:

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C. .. Product synthons were gel purified as described above and split into two equal volumes, which were treated with either BsaI or BsmBI to yield orthogonally protected dimers.

    Article Title: StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins
    Article Snippet: More generally, many proteins tolerate the insertion of protein domains into exposed loops , and should thus be amenable to drug regulation via internal StaPL module insertion and linkage preservation by NS3 protease inhibitors. .. Plasmids were constructed using standard molecular biology techniques: restriction enzyme digest (Fermentas), PCR and overlap extension PCR with PrimeSTAR polymerase (Clontech), and assembly using either In-Fusion enzyme (Clontech) or T4 ligase (Thermo Fisher). .. DNA was transformed into XL10 Gold (Agilent) or Stellar (Clontech) competent E. coli cells with ampicillin (100 μg/mL) selection.

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T-Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a-Latroeggtoxin-V were transformed into BL21 (DE3). .. The transformed strains were plated on LB solid medium containing 100 μg/mL Kanamycin, and single colonies was selected for colony PCR detection after culturing at 37 °C for 16 h. The plasmids extracted from the colonies with positive colony PCR results were analyzed by double enzyme digestion and sequencing, and the strains with the correct analysis results were inoculated into the LB liquid medium containing 100 μg/mL Kanamycin and shaken at 37 °C and 225 r/min overnight.

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T- Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a- Latroeggtoxin-V were transformed into BL21 (DE3). .. The transformed strains were plated on LB solid medium containing 100 μg/mL Kanamycin, and single colonies was selected for colony PCR detection after culturing at 37 °C for 16 h. The plasmids extracted from the colonies with positive colony PCR results were analyzed by double enzyme digestion and sequencing, and the strains with the correct analysis results were inoculated into the LB liquid medium containing 100 μg/mL Kanamycin and shaken at 37 °C and 225 r/min overnight.

    Article Title: Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants
    Article Snippet: Restriction enzymes and T4 ligase were purchased from Thermo Fischer Scientific. .. Restriction enzymes and T4 ligase were purchased from Thermo Fischer Scientific.

    Incubation:

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs
    Article Snippet: 5µl of each forward and reverse oligo, 2µl of 10x oligo annealing buffer (100 mM TrisHCL (pH 8); 10 mM EDTA (pH 8); 1 M NaCl) and 8µl of ddH2 O were incubated at 95°C for 4 minutes and afterwards cooled at room temperature for 5-10 minutes. .. The 10nM annealed double-stranded oligo dilution was used as an insert in a standard ligation reaction with T4 ligase (5U, Thermo, Fisher)

    Article Title: GA-Binding Protein and p300 Are Essential Components of a Retinoic Acid-Induced Enhanceosome in Myeloid Cells
    Article Snippet: Approximately 2 μg of the sample was diluted to 0.8 ml in ligation buffer (30 mM Tris HCl, pH 8.0, 10 mM dithiothreitol, and 1 mM ATP) plus Triton X-100 (final concentration, 1%) and incubated 1 h at 37°C. .. Ligation was then performed at 16°C using 30 Weiss units of T4 ligase (Invitrogen) for 4 h. Samples were then treated with proteinase K (final concentration, 100 μg/ml) and incubated overnight at 65°C to reverse cross-links. .. DNA was then ethanol precipitated, and serial dilutions were used for PCR using primers corresponding to the CD18 distal enhancer (5′-GAAGGCAGAGGTGGATGGA-3′) and proximal promoter (5′-TAGAAACCTCAGCTGGAGGC-3′).

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: Usually around 10.5 µg of (13 FNIII)mono synthon were recovered (∼ 85 % yield). .. Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C. .. Adding additional ligase had little effect on ligation efficiency.

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a-Latroeggtoxin-V were transformed into BL21 (DE3). .. Prokaryotic expression was induced by the addition of IPTG (Isopropyl β-d -1-thiogalactopyranoside) to a final concentration of 0.25 mM.

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a- Latroeggtoxin-V were transformed into BL21 (DE3). .. Prokaryotic expression was induced by the addition of IPTG (Isopropyl β- d -1-thiogalactopyranoside) to a final concentration of 0.25 mM.

    Article Title: B Cell Receptor Affinity for Insulin Dictates Autoantigen Acquisition and B Cell Functionality in Autoimmune Diabetes
    Article Snippet: PCR products and empty vector plasmid were incubated with BglII and SalI restriction enzymes (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocols. .. The digested DNA was gel purified and ligated in multiple ratios of vector:insert with T4 ligase (Life Technologies, Carlsbad, CA, USA) overnight.

    Article Title: Identification of a novel, methylation-dependent, RUNX2 regulatory region associated with osteoarthritis risk
    Article Snippet: The double-stranded DNA oligonucleotides were then ligated into the BbsI linearized CRISPR-Cas9 vector, PX462, using T4 ligase (Invitrogen) overnight at 16°C. .. The double-stranded DNA oligonucleotides were then ligated into the BbsI linearized CRISPR-Cas9 vector, PX462, using T4 ligase (Invitrogen) overnight at 16°C.

    Article Title: Esomeprazole Increases Airway Surface Liquid pH in Primary Cystic Fibrosis Epithelial Cells
    Article Snippet: The double stranded oligonucleotides were then ligated into the CRISPR-Cas9 vector, PX462, following BbsI linearization, using T4 ligase (Invitrogen) overnight at 16°C. .. Following validation in the Tc28a2 cells, primary CF hAECs were transfected using an adapted version of a previously published protocol ( ).

    Gel Extraction:

    Article Title: Splice variants of the endonucleases XPF and XPG contain residual DNA repair capabilities and could be a valuable tool for personalized medicine
    Article Snippet: Ligation was performed in a 3:1 (fragment : vector) ratio using a T4 Ligase (Thermo Fisher Scientific, Waltham, MA, USA). .. Ligation was performed in a 3:1 (fragment : vector) ratio using a T4 Ligase (Thermo Fisher Scientific, Waltham, MA, USA).

    Expressing:

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: Paragraph title: 5.3. Prokaryotic Expression of Latroeggtoxin-V ... After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a-Latroeggtoxin-V were transformed into BL21 (DE3).

    Article Title: B Cell Receptor Affinity for Insulin Dictates Autoantigen Acquisition and B Cell Functionality in Autoimmune Diabetes
    Article Snippet: Paragraph title: 2.5. Retroviral Expression of Immunoglobulin Light Chains ... The digested DNA was gel purified and ligated in multiple ratios of vector:insert with T4 ligase (Life Technologies, Carlsbad, CA, USA) overnight.

    Article Title: High Level Expression and Purification of the Clinically Active Antimicrobial Peptide P-113 in Escherichia coli
    Article Snippet: The expression vector pET-28a and host BL21 (DE3) were purchased from Novagen (Merck Millipore, Burlington, MA, USA). .. Restriction enzymes and T4 ligase were purchased from Thermo Scientific (Waltham, MA, USA).

    Article Title: Model‐driven design of a minimal medium for Akkermansia muciniphila confirms mucus adaptation
    Article Snippet: Paragraph title: Enzyme expression and purification ... The DNA was purified again and ligated with 3:1 ratio (insert:vector) using T4 ligase (manufacturers protocol, Thermo Fisher Scientific).

    Modification:

    Article Title: Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants
    Article Snippet: Restriction enzymes and T4 ligase were purchased from Thermo Fischer Scientific. .. Plasmids were purified with GenElute HP Plasmid Miniprep kit (Sigma-Aldrich).

    Transformation Assay:

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T-Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a-Latroeggtoxin-V were transformed into BL21 (DE3). .. The transformed strains were plated on LB solid medium containing 100 μg/mL Kanamycin, and single colonies was selected for colony PCR detection after culturing at 37 °C for 16 h. The plasmids extracted from the colonies with positive colony PCR results were analyzed by double enzyme digestion and sequencing, and the strains with the correct analysis results were inoculated into the LB liquid medium containing 100 μg/mL Kanamycin and shaken at 37 °C and 225 r/min overnight.

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T- Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a- Latroeggtoxin-V were transformed into BL21 (DE3). .. The transformed strains were plated on LB solid medium containing 100 μg/mL Kanamycin, and single colonies was selected for colony PCR detection after culturing at 37 °C for 16 h. The plasmids extracted from the colonies with positive colony PCR results were analyzed by double enzyme digestion and sequencing, and the strains with the correct analysis results were inoculated into the LB liquid medium containing 100 μg/mL Kanamycin and shaken at 37 °C and 225 r/min overnight.

    Article Title: System for the heterologous expression of NS1 protein of H9N2 avian influenza virus in the ciliate Tetrahymena thermophila
    Article Snippet: Paragraph title: Construction of transformation plasmid pD5H8-NS1 ... Then, the inserts and vector were mixed at a molar ratio of 6:1 and ligated overnight at 16°C with T4 ligase (Thermo Fisher Scientific, Waltham, MA, U.S.A.).

    Article Title: Model‐driven design of a minimal medium for Akkermansia muciniphila confirms mucus adaptation
    Article Snippet: The DNA was purified again and ligated with 3:1 ratio (insert:vector) using T4 ligase (manufacturers protocol, Thermo Fisher Scientific). .. A selected colony was grown in 10 ml liquid LB until an OD600 of 1 after which plasmids were purified.

    Derivative Assay:

    Article Title: StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins
    Article Snippet: Plasmids were constructed using standard molecular biology techniques: restriction enzyme digest (Fermentas), PCR and overlap extension PCR with PrimeSTAR polymerase (Clontech), and assembly using either In-Fusion enzyme (Clontech) or T4 ligase (Thermo Fisher). .. Subcloned regions were verified by Sanger sequencing, assisted by Geneious software (Biomatters).

    Gel Purification:

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: After a 30 min gel electrophoresis run on 2% (w/v) agarose gels, synthon fragments were excised and gel purified using a DNA gel purification kit (Promega). .. Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C.

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T-Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a-Latroeggtoxin-V were transformed into BL21 (DE3). .. The transformed strains were plated on LB solid medium containing 100 μg/mL Kanamycin, and single colonies was selected for colony PCR detection after culturing at 37 °C for 16 h. The plasmids extracted from the colonies with positive colony PCR results were analyzed by double enzyme digestion and sequencing, and the strains with the correct analysis results were inoculated into the LB liquid medium containing 100 μg/mL Kanamycin and shaken at 37 °C and 225 r/min overnight.

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T- Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a- Latroeggtoxin-V were transformed into BL21 (DE3). .. The transformed strains were plated on LB solid medium containing 100 μg/mL Kanamycin, and single colonies was selected for colony PCR detection after culturing at 37 °C for 16 h. The plasmids extracted from the colonies with positive colony PCR results were analyzed by double enzyme digestion and sequencing, and the strains with the correct analysis results were inoculated into the LB liquid medium containing 100 μg/mL Kanamycin and shaken at 37 °C and 225 r/min overnight.

    Transfection:

    Article Title: Esomeprazole Increases Airway Surface Liquid pH in Primary Cystic Fibrosis Epithelial Cells
    Article Snippet: The double stranded oligonucleotides were then ligated into the CRISPR-Cas9 vector, PX462, following BbsI linearization, using T4 ligase (Invitrogen) overnight at 16°C. .. Deletion of the target region was confirmed using end-point PCR (Supplementary Figure ).

    Gas Chromatography:

    Article Title: FTO Knockout Causes Chromosome Instability and G2/M Arrest in Mouse GC-1 Cells
    Article Snippet: For knocking out FTO in GC-1 cells, the following sgRNAs were designed and synthesized, sg-FTO1U: 5′-ACCGCCGTCCTGCGATGATGAAG-3′, sg-FTO1D: 5′-AAACCTTCATCATCGCAGGACGG-3′, sg-FTO2U: 5′-ACCGGAACTCTGCCATGCACAG-3′, sg-FTO2D: 5′-AAACCTGTGCATGGCAGAGTTC-3′. .. Plasmid was ligated with annealed sgRNAs via T4 ligase (Thermo Fisher Scientific).

    Ligation:

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs
    Article Snippet: 1µl of annealed double-stranded oligo mixture was diluted in 5µl 10x oligo annealing buffer and 44µl ddH2O to obtain a final concentration of 10nM. .. The 10nM annealed double-stranded oligo dilution was used as an insert in a standard ligation reaction with T4 ligase (5U, Thermo, Fisher) .. Reactions were set up using 20 fmol of each Golden Gate entry vector and the destination vector, 10 U of BsaI Restriction Enzyme (Thermo, Fisher Fast Digest Enzyme Eco31I) and 30 U of T4 DNA Ligase (Thermo, Fisher) in 2 µl 10x ligation buffer (10x ligation buffer was prepared by supplementing the Thermo, Fisher FastDigest buffer with 10mM dATP and 100mM DTT) to a final reaction volume of 20 µl with ddH2O.

    Article Title: Chromosome conformation capture of transcriptional interactions between cytochrome c oxidase genes and genes of glutamatergic synaptic transmission in neurons
    Article Snippet: Afterwards, restriction enzyme was heat-inactivated and chromatin was diluted in T4 ligation buffer to achieve a low genomic DNA concentration of 2.5 ng/μl to facilitate intermolecular re-ligation. .. Chromatin re-ligation was performed by incubating diluted chromatin with 100 Weiss units of T4 ligase (Fermentas) for 4 h at 16°C.

    Article Title: GA-Binding Protein and p300 Are Essential Components of a Retinoic Acid-Induced Enhanceosome in Myeloid Cells
    Article Snippet: Approximately 2 μg of the sample was diluted to 0.8 ml in ligation buffer (30 mM Tris HCl, pH 8.0, 10 mM dithiothreitol, and 1 mM ATP) plus Triton X-100 (final concentration, 1%) and incubated 1 h at 37°C. .. Ligation was then performed at 16°C using 30 Weiss units of T4 ligase (Invitrogen) for 4 h. Samples were then treated with proteinase K (final concentration, 100 μg/ml) and incubated overnight at 65°C to reverse cross-links. .. DNA was then ethanol precipitated, and serial dilutions were used for PCR using primers corresponding to the CD18 distal enhancer (5′-GAAGGCAGAGGTGGATGGA-3′) and proximal promoter (5′-TAGAAACCTCAGCTGGAGGC-3′).

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: Usually around 10.5 µg of (13 FNIII)mono synthon were recovered (∼ 85 % yield). .. Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C. .. Adding additional ligase had little effect on ligation efficiency.

    Article Title: Splice variants of the endonucleases XPF and XPG contain residual DNA repair capabilities and could be a valuable tool for personalized medicine
    Article Snippet: Amplification fragments as well as the target vector were digested with appropriate restriction enzymes (listed in Table ) according to manufacturer’s protocol (NEB, Ipswich, MA, USA). .. Ligation was performed in a 3:1 (fragment : vector) ratio using a T4 Ligase (Thermo Fisher Scientific, Waltham, MA, USA). .. Constructs were transformed in chemically competent DH5α (E.coli) cells and isolated from single clones grown on LB Agar plates using the NucleoBond Mini plasmid kit (Machery + Nagel, Düren, DE).

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T-Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a-Latroeggtoxin-V were transformed into BL21 (DE3). .. The transformed strains were plated on LB solid medium containing 100 μg/mL Kanamycin, and single colonies was selected for colony PCR detection after culturing at 37 °C for 16 h. The plasmids extracted from the colonies with positive colony PCR results were analyzed by double enzyme digestion and sequencing, and the strains with the correct analysis results were inoculated into the LB liquid medium containing 100 μg/mL Kanamycin and shaken at 37 °C and 225 r/min overnight.

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T- Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a- Latroeggtoxin-V were transformed into BL21 (DE3). .. The transformed strains were plated on LB solid medium containing 100 μg/mL Kanamycin, and single colonies was selected for colony PCR detection after culturing at 37 °C for 16 h. The plasmids extracted from the colonies with positive colony PCR results were analyzed by double enzyme digestion and sequencing, and the strains with the correct analysis results were inoculated into the LB liquid medium containing 100 μg/mL Kanamycin and shaken at 37 °C and 225 r/min overnight.

    Article Title: FTO Knockout Causes Chromosome Instability and G2/M Arrest in Mouse GC-1 Cells
    Article Snippet: Plasmid was ligated with annealed sgRNAs via T4 ligase (Thermo Fisher Scientific). .. PCR products were purified by the PCR clean-up Kit (Axgen).

    Article Title: System for the heterologous expression of NS1 protein of H9N2 avian influenza virus in the ciliate Tetrahymena thermophila
    Article Snippet: Then, the inserts and vector were mixed at a molar ratio of 6:1 and ligated overnight at 16°C with T4 ligase (Thermo Fisher Scientific, Waltham, MA, U.S.A.). .. Then, the inserts and vector were mixed at a molar ratio of 6:1 and ligated overnight at 16°C with T4 ligase (Thermo Fisher Scientific, Waltham, MA, U.S.A.).

    Synthesized:

    Article Title: FTO Knockout Causes Chromosome Instability and G2/M Arrest in Mouse GC-1 Cells
    Article Snippet: For knocking out FTO in GC-1 cells, the following sgRNAs were designed and synthesized, sg-FTO1U: 5′-ACCGCCGTCCTGCGATGATGAAG-3′, sg-FTO1D: 5′-AAACCTTCATCATCGCAGGACGG-3′, sg-FTO2U: 5′-ACCGGAACTCTGCCATGCACAG-3′, sg-FTO2D: 5′-AAACCTGTGCATGGCAGAGTTC-3′. .. Plasmid was ligated with annealed sgRNAs via T4 ligase (Thermo Fisher Scientific).

    Cell Culture:

    Article Title: Chromosome conformation capture of transcriptional interactions between cytochrome c oxidase genes and genes of glutamatergic synaptic transmission in neurons
    Article Snippet: To cross-link chromatin, 1 × 107 N2a cells, 5 × 106 primary neurons, or 1 × 107 C2C12 cells in 10 ml of cell culture media were treated with 2% formaldehyde for 10 min at room temperature (RT). .. Chromatin re-ligation was performed by incubating diluted chromatin with 100 Weiss units of T4 ligase (Fermentas) for 4 h at 16°C.

    other:

    Article Title: DNAzyme-based probe for circulating microRNA detection in peripheral blood
    Article Snippet: For miRNA detection, 2 μL miR-105 was mixed with 3 μL 10× reaction buffer (330 mM Tris-acetate, 100 mM magnesium acetate [Mg(Ac)2 ], 660 mM potassium acetate [KAc], 1% Tween-20, and 10 mM dithiothreitol [DTT] [pH 7.9], 2 μL [10 U/μL] T4 ligase [Fermentas], 0.5 μL 10 μM deoxy-nucleotide triphosphates [dNTPs] mixture [Takara, Dalian, People’s Republic of China], and RNase-free water), 25 μL double distilled water.

    Sequencing:

    Article Title: StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins
    Article Snippet: Plasmids were constructed using standard molecular biology techniques: restriction enzyme digest (Fermentas), PCR and overlap extension PCR with PrimeSTAR polymerase (Clontech), and assembly using either In-Fusion enzyme (Clontech) or T4 ligase (Thermo Fisher). .. Plasmid DNA was isolated with the PureLink hiPure Plasmid Maxiprep Kit (Thermo Fisher) or the Plasmid Plus Midiprep Kit (Qiagen).

    Article Title: Splice variants of the endonucleases XPF and XPG contain residual DNA repair capabilities and could be a valuable tool for personalized medicine
    Article Snippet: Ligation was performed in a 3:1 (fragment : vector) ratio using a T4 Ligase (Thermo Fisher Scientific, Waltham, MA, USA). .. Constructs were transformed in chemically competent DH5α (E.coli) cells and isolated from single clones grown on LB Agar plates using the NucleoBond Mini plasmid kit (Machery + Nagel, Düren, DE).

    Article Title: Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants
    Article Snippet: Restriction enzymes and T4 ligase were purchased from Thermo Fischer Scientific. .. Restriction enzymes and T4 ligase were purchased from Thermo Fischer Scientific.

    Article Title: B Cell Receptor Affinity for Insulin Dictates Autoantigen Acquisition and B Cell Functionality in Autoimmune Diabetes
    Article Snippet: For directional cloning, a 5′ primer was designed with a BglII site before the Kozak sequence and 3′ with SalI after the stop codon: Forward 5′-AATAGATCTGCCACCATGGGATGGTCATGTATCATCC-3′, Reverse 5′-TATGTCGACCTAACACTCTCCCCTGTTGAAGCTC-3′. .. The digested DNA was gel purified and ligated in multiple ratios of vector:insert with T4 ligase (Life Technologies, Carlsbad, CA, USA) overnight.

    Article Title: Identification of a novel, methylation-dependent, RUNX2 regulatory region associated with osteoarthritis risk
    Article Snippet: Single-stranded DNA oligonucleotides (Sigma Aldrich) containing the desired gRNA sequence along with the BbsI recognition motif were annealed with the reverse complementary strand (95°C–25°C, Δ-6°C/min). .. The double-stranded DNA oligonucleotides were then ligated into the BbsI linearized CRISPR-Cas9 vector, PX462, using T4 ligase (Invitrogen) overnight at 16°C.

    Article Title: A genetically inducible porcine model of intestinal cancer
    Article Snippet: In brief, gDNA isolated from fetal TG fibroblasts was digested with Bsu 36I and Pac I (Thermo Fisher). .. DNA was circularized by T4 ligase (Thermo Fisher) and used as template in a two‐round nested PCR setup to produce flanking genomic sequence. .. The DNA was Sanger‐sequenced and the upstream genomic sequence was mapped to the pig genome (Groenen et al ., ) (S. scrofa 10.2).

    Article Title: Esomeprazole Increases Airway Surface Liquid pH in Primary Cystic Fibrosis Epithelial Cells
    Article Snippet: Single stranded DNA oligonucleotides (IDT) containing the gRNA sequence in addition to the BbsI restriction enzyme sequences were annealed (95 to 25°C, Δ-6°C/min). .. The double stranded oligonucleotides were then ligated into the CRISPR-Cas9 vector, PX462, following BbsI linearization, using T4 ligase (Invitrogen) overnight at 16°C.

    Article Title: Model‐driven design of a minimal medium for Akkermansia muciniphila confirms mucus adaptation
    Article Snippet: The A. muciniphila gene NagB on locus Amuc_1822 (AMUC_RS09725) was amplified using the forward primer 5′‐ATAGA GGTACC ATGATCGGGGTGGAAAGT‐3′ and the reverse primer 5′‐ TATGC CCTAGG TTA ATGGTGGTGGTGATGATG AAGGAGGGAAGCAGCCC‐3′, adding a C‐terminal His‐tag (bold in primer sequence) and restriction sites (underlined in primer sequence). .. The DNA was purified again and ligated with 3:1 ratio (insert:vector) using T4 ligase (manufacturers protocol, Thermo Fisher Scientific).

    Recombinant:

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T-Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a-Latroeggtoxin-V were transformed into BL21 (DE3). .. The transformed strains were plated on LB solid medium containing 100 μg/mL Kanamycin, and single colonies was selected for colony PCR detection after culturing at 37 °C for 16 h. The plasmids extracted from the colonies with positive colony PCR results were analyzed by double enzyme digestion and sequencing, and the strains with the correct analysis results were inoculated into the LB liquid medium containing 100 μg/mL Kanamycin and shaken at 37 °C and 225 r/min overnight.

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T- Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a- Latroeggtoxin-V were transformed into BL21 (DE3). .. The transformed strains were plated on LB solid medium containing 100 μg/mL Kanamycin, and single colonies was selected for colony PCR detection after culturing at 37 °C for 16 h. The plasmids extracted from the colonies with positive colony PCR results were analyzed by double enzyme digestion and sequencing, and the strains with the correct analysis results were inoculated into the LB liquid medium containing 100 μg/mL Kanamycin and shaken at 37 °C and 225 r/min overnight.

    Nucleic Acid Electrophoresis:

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: After a 30 min gel electrophoresis run on 2% (w/v) agarose gels, synthon fragments were excised and gel purified using a DNA gel purification kit (Promega). .. Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C.

    Isolation:

    Article Title: Chromosome conformation capture of transcriptional interactions between cytochrome c oxidase genes and genes of glutamatergic synaptic transmission in neurons
    Article Snippet: Nuclei were isolated by incubating cells in a lysis buffer (10 mM Tris, pH 8.0, 10 mM NaCl, 0.2% NP-40) on ice with agitation for 30 min. Chromatin was subsequently released by treating isolated nuclei with 0.3% SDS and digested with 400 units of Bgl II (Fermentas, Hanover, MD) overnight. .. Chromatin re-ligation was performed by incubating diluted chromatin with 100 Weiss units of T4 ligase (Fermentas) for 4 h at 16°C.

    Article Title: StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins
    Article Snippet: Plasmids were constructed using standard molecular biology techniques: restriction enzyme digest (Fermentas), PCR and overlap extension PCR with PrimeSTAR polymerase (Clontech), and assembly using either In-Fusion enzyme (Clontech) or T4 ligase (Thermo Fisher). .. DNA was transformed into XL10 Gold (Agilent) or Stellar (Clontech) competent E. coli cells with ampicillin (100 μg/mL) selection.

    Article Title: Identification of a novel, methylation-dependent, RUNX2 regulatory region associated with osteoarthritis risk
    Article Snippet: The double-stranded DNA oligonucleotides were then ligated into the BbsI linearized CRISPR-Cas9 vector, PX462, using T4 ligase (Invitrogen) overnight at 16°C. .. For nucleofection, 106 cells per biological replicate were incubated with 5 μg of plasmid DNA using the manufacturer-recommended Cell Line 4D-Nucleofector X Kit in combination with the 4D-Nucleofector System (Lonza).

    Article Title: A genetically inducible porcine model of intestinal cancer
    Article Snippet: In brief, gDNA isolated from fetal TG fibroblasts was digested with Bsu 36I and Pac I (Thermo Fisher). .. DNA was circularized by T4 ligase (Thermo Fisher) and used as template in a two‐round nested PCR setup to produce flanking genomic sequence.

    Labeling:

    Article Title: High Level Expression and Purification of the Clinically Active Antimicrobial Peptide P-113 in Escherichia coli
    Article Snippet: Restriction enzymes and T4 ligase were purchased from Thermo Scientific (Waltham, MA, USA). .. Restriction enzymes and T4 ligase were purchased from Thermo Scientific (Waltham, MA, USA).

    Purification:

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: After a 30 min gel electrophoresis run on 2% (w/v) agarose gels, synthon fragments were excised and gel purified using a DNA gel purification kit (Promega). .. Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C.

    Article Title: Splice variants of the endonucleases XPF and XPG contain residual DNA repair capabilities and could be a valuable tool for personalized medicine
    Article Snippet: Restriction sites for specific restriction enzymes were added by a second PCR reaction of the purified fragments and afterwards precipitated with ethanol under high salt conditions. .. Ligation was performed in a 3:1 (fragment : vector) ratio using a T4 Ligase (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants
    Article Snippet: Restriction enzymes and T4 ligase were purchased from Thermo Fischer Scientific. .. All DNA constructs were verified by Sanger sequencing (Microsynth AG).

    Article Title: FTO Knockout Causes Chromosome Instability and G2/M Arrest in Mouse GC-1 Cells
    Article Snippet: Plasmid was ligated with annealed sgRNAs via T4 ligase (Thermo Fisher Scientific). .. The following primers were designed and synthesized for the amplification of FTO CDS, FTO-res-F: 5′-GAATCTAGAATGAAGCGCGTCCAGAC-3′, FTO-res-R: 5′-GGAGAATTCTGCTGGAAGCAAGATCCTAG-3′.

    Article Title: B Cell Receptor Affinity for Insulin Dictates Autoantigen Acquisition and B Cell Functionality in Autoimmune Diabetes
    Article Snippet: PCR products and empty vector plasmid were incubated with BglII and SalI restriction enzymes (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocols. .. The digested DNA was gel purified and ligated in multiple ratios of vector:insert with T4 ligase (Life Technologies, Carlsbad, CA, USA) overnight. .. Ligation mixtures were used to transform Stbl2 competent cells (Life Technologies, Carlsbad, CA, USA) per the manufacturer’s protocols, and transformed cells were plated overnight on lysogeny broth (LB) agar plates + 50 μg/mL ampicillin (Sigma, St. Louis, MO, USA) at 30 °C.

    Article Title: Model‐driven design of a minimal medium for Akkermansia muciniphila confirms mucus adaptation
    Article Snippet: The purified amplicon and vector pCDF‐1b (Novagen, Merck Milipore, Darmstadt, Germany) were digested using enzymes XmaIJ (Thermo Fisher Scientific) and KpnI HF (New England Biolabs) in Tango buffer (Thermo Fisher Scientific) for 1.5 h at 37°C. .. The DNA was purified again and ligated with 3:1 ratio (insert:vector) using T4 ligase (manufacturers protocol, Thermo Fisher Scientific). .. Competent Escherichia coli DH10B (manufacturers protocol, New England Biolabs) were transformed with 2 μl ligation mixture and plated on LB agar with 50 ug/ml spectinomycin.

    Polymerase Chain Reaction:

    Article Title: StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins
    Article Snippet: More generally, many proteins tolerate the insertion of protein domains into exposed loops , and should thus be amenable to drug regulation via internal StaPL module insertion and linkage preservation by NS3 protease inhibitors. .. Plasmids were constructed using standard molecular biology techniques: restriction enzyme digest (Fermentas), PCR and overlap extension PCR with PrimeSTAR polymerase (Clontech), and assembly using either In-Fusion enzyme (Clontech) or T4 ligase (Thermo Fisher). .. DNA was transformed into XL10 Gold (Agilent) or Stellar (Clontech) competent E. coli cells with ampicillin (100 μg/mL) selection.

    Article Title: Splice variants of the endonucleases XPF and XPG contain residual DNA repair capabilities and could be a valuable tool for personalized medicine
    Article Snippet: Restriction sites for specific restriction enzymes were added by a second PCR reaction of the purified fragments and afterwards precipitated with ethanol under high salt conditions. .. Ligation was performed in a 3:1 (fragment : vector) ratio using a T4 Ligase (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: FTO Knockout Causes Chromosome Instability and G2/M Arrest in Mouse GC-1 Cells
    Article Snippet: Plasmid was ligated with annealed sgRNAs via T4 ligase (Thermo Fisher Scientific). .. The following primers were designed and synthesized for the amplification of FTO CDS, FTO-res-F: 5′-GAATCTAGAATGAAGCGCGTCCAGAC-3′, FTO-res-R: 5′-GGAGAATTCTGCTGGAAGCAAGATCCTAG-3′.

    Article Title: B Cell Receptor Affinity for Insulin Dictates Autoantigen Acquisition and B Cell Functionality in Autoimmune Diabetes
    Article Snippet: PCR products and empty vector plasmid were incubated with BglII and SalI restriction enzymes (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocols. .. The digested DNA was gel purified and ligated in multiple ratios of vector:insert with T4 ligase (Life Technologies, Carlsbad, CA, USA) overnight.

    Article Title: Identification of a novel, methylation-dependent, RUNX2 regulatory region associated with osteoarthritis risk
    Article Snippet: The double-stranded DNA oligonucleotides were then ligated into the BbsI linearized CRISPR-Cas9 vector, PX462, using T4 ligase (Invitrogen) overnight at 16°C. .. Following nucleofection, cell monolayers were grown in each well of a six well plate and nucleofected cells were selected with puromycin after 24 h. Following expansion of cells into T25 culture flasks, nucleic acids were extracted using the EZNA DNA/RNA Isolation kit (Omega Bio-Tek) according to the manufacturer’s protocol.

    Article Title: A genetically inducible porcine model of intestinal cancer
    Article Snippet: Paragraph title: Long‐distance inverted PCR (LDI‐PCR) ... DNA was circularized by T4 ligase (Thermo Fisher) and used as template in a two‐round nested PCR setup to produce flanking genomic sequence.

    Article Title: System for the heterologous expression of NS1 protein of H9N2 avian influenza virus in the ciliate Tetrahymena thermophila
    Article Snippet: The cassette structure in pTIEV4 vector is shown in The PCR products and pTIEV4 vector were digested with Bam HI andXho I (New England Biolabs, Ipswich, MA, U.S.A.) separately. .. Then, the inserts and vector were mixed at a molar ratio of 6:1 and ligated overnight at 16°C with T4 ligase (Thermo Fisher Scientific, Waltham, MA, U.S.A.).

    Article Title: Esomeprazole Increases Airway Surface Liquid pH in Primary Cystic Fibrosis Epithelial Cells
    Article Snippet: The double stranded oligonucleotides were then ligated into the CRISPR-Cas9 vector, PX462, following BbsI linearization, using T4 ligase (Invitrogen) overnight at 16°C. .. To validate the sequences, cells from the human chondrocyte cell line Tc28a2 were nucleofected [106 cells, 5 μg of plasmid DNA using the manufacturer recommended Cell Line 4D-Nucleofector X Kit in combination with the 4D-Nucleofector System (Lonza)] and selected with puromycin after 24 h. Following expansion, nucleic acids were extracted using the EZNA DNA/RNA Isolation kit (Omega Bio-Tek) according to the manufacturer’s protocol.

    Cotransfection:

    Article Title: B Cell Receptor Affinity for Insulin Dictates Autoantigen Acquisition and B Cell Functionality in Autoimmune Diabetes
    Article Snippet: The digested DNA was gel purified and ligated in multiple ratios of vector:insert with T4 ligase (Life Technologies, Carlsbad, CA, USA) overnight. .. Plasmids were purified from overnight cultures using Miniprep (Qiagen, Valencia, CA, USA) and insert verified by restriction enzyme digestion, followed by sequencing.

    CRISPR:

    Article Title: Identification of a novel, methylation-dependent, RUNX2 regulatory region associated with osteoarthritis risk
    Article Snippet: Single-stranded DNA oligonucleotides (Sigma Aldrich) containing the desired gRNA sequence along with the BbsI recognition motif were annealed with the reverse complementary strand (95°C–25°C, Δ-6°C/min). .. The double-stranded DNA oligonucleotides were then ligated into the BbsI linearized CRISPR-Cas9 vector, PX462, using T4 ligase (Invitrogen) overnight at 16°C. .. Cells from the human chondrocyte cell line Tc28a2 were grown in monolayer culture as previously described ( ) to 70–80% confluence.

    Article Title: Esomeprazole Increases Airway Surface Liquid pH in Primary Cystic Fibrosis Epithelial Cells
    Article Snippet: Single stranded DNA oligonucleotides (IDT) containing the gRNA sequence in addition to the BbsI restriction enzyme sequences were annealed (95 to 25°C, Δ-6°C/min). .. The double stranded oligonucleotides were then ligated into the CRISPR-Cas9 vector, PX462, following BbsI linearization, using T4 ligase (Invitrogen) overnight at 16°C. .. To validate the sequences, cells from the human chondrocyte cell line Tc28a2 were nucleofected [106 cells, 5 μg of plasmid DNA using the manufacturer recommended Cell Line 4D-Nucleofector X Kit in combination with the 4D-Nucleofector System (Lonza)] and selected with puromycin after 24 h. Following expansion, nucleic acids were extracted using the EZNA DNA/RNA Isolation kit (Omega Bio-Tek) according to the manufacturer’s protocol.

    Nested PCR:

    Article Title: A genetically inducible porcine model of intestinal cancer
    Article Snippet: In brief, gDNA isolated from fetal TG fibroblasts was digested with Bsu 36I and Pac I (Thermo Fisher). .. DNA was circularized by T4 ligase (Thermo Fisher) and used as template in a two‐round nested PCR setup to produce flanking genomic sequence. .. The DNA was Sanger‐sequenced and the upstream genomic sequence was mapped to the pig genome (Groenen et al ., ) (S. scrofa 10.2).

    SDS Page:

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a-Latroeggtoxin-V were transformed into BL21 (DE3). .. Prokaryotic expression was induced by the addition of IPTG (Isopropyl β-d -1-thiogalactopyranoside) to a final concentration of 0.25 mM.

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a- Latroeggtoxin-V were transformed into BL21 (DE3). .. Prokaryotic expression was induced by the addition of IPTG (Isopropyl β- d -1-thiogalactopyranoside) to a final concentration of 0.25 mM.

    Plasmid Preparation:

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: Plasmid DNA of pCR8-13 FNIII and pCR8-VWFA2 was prepared from 50 mL o/n cultures using a Midiprep kit (Sigma Aldrich) and concentrated by ethanol precipitation to obtain ∼0.7 mg DNA at 1.5–2.5 µg/µl. .. Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C.

    Article Title: StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins
    Article Snippet: Plasmids were constructed using standard molecular biology techniques: restriction enzyme digest (Fermentas), PCR and overlap extension PCR with PrimeSTAR polymerase (Clontech), and assembly using either In-Fusion enzyme (Clontech) or T4 ligase (Thermo Fisher). .. DNA was transformed into XL10 Gold (Agilent) or Stellar (Clontech) competent E. coli cells with ampicillin (100 μg/mL) selection.

    Article Title: Splice variants of the endonucleases XPF and XPG contain residual DNA repair capabilities and could be a valuable tool for personalized medicine
    Article Snippet: Amplification fragments as well as the target vector were digested with appropriate restriction enzymes (listed in Table ) according to manufacturer’s protocol (NEB, Ipswich, MA, USA). .. Ligation was performed in a 3:1 (fragment : vector) ratio using a T4 Ligase (Thermo Fisher Scientific, Waltham, MA, USA). .. Constructs were transformed in chemically competent DH5α (E.coli) cells and isolated from single clones grown on LB Agar plates using the NucleoBond Mini plasmid kit (Machery + Nagel, Düren, DE).

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T-Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a-Latroeggtoxin-V were transformed into BL21 (DE3).

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T- Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a- Latroeggtoxin-V were transformed into BL21 (DE3).

    Article Title: Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants
    Article Snippet: Restriction enzymes and T4 ligase were purchased from Thermo Fischer Scientific. .. All DNA constructs were verified by Sanger sequencing (Microsynth AG).

    Article Title: FTO Knockout Causes Chromosome Instability and G2/M Arrest in Mouse GC-1 Cells
    Article Snippet: The PGL3-U6-PGK plasmid (gifted from Shanghai Tech University) was used as the backbone. .. Plasmid was ligated with annealed sgRNAs via T4 ligase (Thermo Fisher Scientific). .. For the FTO rescue experiment, total RNA was extracted from GC-1 cells using RNAiso plus Reagent (Takara Clontech). cDNA was synthesized by the first strand cDNA synthesis kit (Takara Clontech) following the manufacturer’s instructions.

    Article Title: B Cell Receptor Affinity for Insulin Dictates Autoantigen Acquisition and B Cell Functionality in Autoimmune Diabetes
    Article Snippet: PCR products and empty vector plasmid were incubated with BglII and SalI restriction enzymes (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocols. .. The digested DNA was gel purified and ligated in multiple ratios of vector:insert with T4 ligase (Life Technologies, Carlsbad, CA, USA) overnight. .. Ligation mixtures were used to transform Stbl2 competent cells (Life Technologies, Carlsbad, CA, USA) per the manufacturer’s protocols, and transformed cells were plated overnight on lysogeny broth (LB) agar plates + 50 μg/mL ampicillin (Sigma, St. Louis, MO, USA) at 30 °C.

    Article Title: Identification of a novel, methylation-dependent, RUNX2 regulatory region associated with osteoarthritis risk
    Article Snippet: Single-stranded DNA oligonucleotides (Sigma Aldrich) containing the desired gRNA sequence along with the BbsI recognition motif were annealed with the reverse complementary strand (95°C–25°C, Δ-6°C/min). .. The double-stranded DNA oligonucleotides were then ligated into the BbsI linearized CRISPR-Cas9 vector, PX462, using T4 ligase (Invitrogen) overnight at 16°C. .. Cells from the human chondrocyte cell line Tc28a2 were grown in monolayer culture as previously described ( ) to 70–80% confluence.

    Article Title: System for the heterologous expression of NS1 protein of H9N2 avian influenza virus in the ciliate Tetrahymena thermophila
    Article Snippet: The cassette structure in pTIEV4 vector is shown in The PCR products and pTIEV4 vector were digested with Bam HI andXho I (New England Biolabs, Ipswich, MA, U.S.A.) separately. .. Then, the inserts and vector were mixed at a molar ratio of 6:1 and ligated overnight at 16°C with T4 ligase (Thermo Fisher Scientific, Waltham, MA, U.S.A.). .. The ligation products were transformed into competent E. coli DH5α cells and grown in Luria Bertani agar medium containing ampicillin 100 µ g/ml . pTIEV4-NS1-positive colonies were identified by PCR using forward (pTIEV4-1206F) and reverse primers (pTIEV4-1496R) ( ).

    Article Title: High Level Expression and Purification of the Clinically Active Antimicrobial Peptide P-113 in Escherichia coli
    Article Snippet: The expression vector pET-28a and host BL21 (DE3) were purchased from Novagen (Merck Millipore, Burlington, MA, USA). .. Restriction enzymes and T4 ligase were purchased from Thermo Scientific (Waltham, MA, USA).

    Article Title: Esomeprazole Increases Airway Surface Liquid pH in Primary Cystic Fibrosis Epithelial Cells
    Article Snippet: Single stranded DNA oligonucleotides (IDT) containing the gRNA sequence in addition to the BbsI restriction enzyme sequences were annealed (95 to 25°C, Δ-6°C/min). .. The double stranded oligonucleotides were then ligated into the CRISPR-Cas9 vector, PX462, following BbsI linearization, using T4 ligase (Invitrogen) overnight at 16°C. .. To validate the sequences, cells from the human chondrocyte cell line Tc28a2 were nucleofected [106 cells, 5 μg of plasmid DNA using the manufacturer recommended Cell Line 4D-Nucleofector X Kit in combination with the 4D-Nucleofector System (Lonza)] and selected with puromycin after 24 h. Following expansion, nucleic acids were extracted using the EZNA DNA/RNA Isolation kit (Omega Bio-Tek) according to the manufacturer’s protocol.

    Article Title: Model‐driven design of a minimal medium for Akkermansia muciniphila confirms mucus adaptation
    Article Snippet: The purified amplicon and vector pCDF‐1b (Novagen, Merck Milipore, Darmstadt, Germany) were digested using enzymes XmaIJ (Thermo Fisher Scientific) and KpnI HF (New England Biolabs) in Tango buffer (Thermo Fisher Scientific) for 1.5 h at 37°C. .. The DNA was purified again and ligated with 3:1 ratio (insert:vector) using T4 ligase (manufacturers protocol, Thermo Fisher Scientific).

    Software:

    Article Title: StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins
    Article Snippet: Plasmids were constructed using standard molecular biology techniques: restriction enzyme digest (Fermentas), PCR and overlap extension PCR with PrimeSTAR polymerase (Clontech), and assembly using either In-Fusion enzyme (Clontech) or T4 ligase (Thermo Fisher). .. Plasmid DNA was isolated with the PureLink hiPure Plasmid Maxiprep Kit (Thermo Fisher) or the Plasmid Plus Midiprep Kit (Qiagen).

    Positron Emission Tomography:

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T-Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a-Latroeggtoxin-V were transformed into BL21 (DE3). .. The transformed strains were plated on LB solid medium containing 100 μg/mL Kanamycin, and single colonies was selected for colony PCR detection after culturing at 37 °C for 16 h. The plasmids extracted from the colonies with positive colony PCR results were analyzed by double enzyme digestion and sequencing, and the strains with the correct analysis results were inoculated into the LB liquid medium containing 100 μg/mL Kanamycin and shaken at 37 °C and 225 r/min overnight.

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: The pMD19-T- Latroeggtoxin-V plasmids were extracted by a plasmid extraction kit (TIANGEN, Beijing, China) from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI (Thermo Fisher Scientific, Waltham, MA, USA). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a- Latroeggtoxin-V were transformed into BL21 (DE3). .. The transformed strains were plated on LB solid medium containing 100 μg/mL Kanamycin, and single colonies was selected for colony PCR detection after culturing at 37 °C for 16 h. The plasmids extracted from the colonies with positive colony PCR results were analyzed by double enzyme digestion and sequencing, and the strains with the correct analysis results were inoculated into the LB liquid medium containing 100 μg/mL Kanamycin and shaken at 37 °C and 225 r/min overnight.

    Article Title: High Level Expression and Purification of the Clinically Active Antimicrobial Peptide P-113 in Escherichia coli
    Article Snippet: The expression vector pET-28a and host BL21 (DE3) were purchased from Novagen (Merck Millipore, Burlington, MA, USA). .. Restriction enzymes and T4 ligase were purchased from Thermo Scientific (Waltham, MA, USA).

    Ethanol Precipitation:

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: Plasmid DNA of pCR8-13 FNIII and pCR8-VWFA2 was prepared from 50 mL o/n cultures using a Midiprep kit (Sigma Aldrich) and concentrated by ethanol precipitation to obtain ∼0.7 mg DNA at 1.5–2.5 µg/µl. .. Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C.

    Concentration Assay:

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs
    Article Snippet: 1µl of annealed double-stranded oligo mixture was diluted in 5µl 10x oligo annealing buffer and 44µl ddH2O to obtain a final concentration of 10nM. .. The 10nM annealed double-stranded oligo dilution was used as an insert in a standard ligation reaction with T4 ligase (5U, Thermo, Fisher)

    Article Title: Chromosome conformation capture of transcriptional interactions between cytochrome c oxidase genes and genes of glutamatergic synaptic transmission in neurons
    Article Snippet: Afterwards, restriction enzyme was heat-inactivated and chromatin was diluted in T4 ligation buffer to achieve a low genomic DNA concentration of 2.5 ng/μl to facilitate intermolecular re-ligation. .. Chromatin re-ligation was performed by incubating diluted chromatin with 100 Weiss units of T4 ligase (Fermentas) for 4 h at 16°C.

    Article Title: GA-Binding Protein and p300 Are Essential Components of a Retinoic Acid-Induced Enhanceosome in Myeloid Cells
    Article Snippet: Approximately 2 μg of the sample was diluted to 0.8 ml in ligation buffer (30 mM Tris HCl, pH 8.0, 10 mM dithiothreitol, and 1 mM ATP) plus Triton X-100 (final concentration, 1%) and incubated 1 h at 37°C. .. Ligation was then performed at 16°C using 30 Weiss units of T4 ligase (Invitrogen) for 4 h. Samples were then treated with proteinase K (final concentration, 100 μg/ml) and incubated overnight at 65°C to reverse cross-links. .. DNA was then ethanol precipitated, and serial dilutions were used for PCR using primers corresponding to the CD18 distal enhancer (5′-GAAGGCAGAGGTGGATGGA-3′) and proximal promoter (5′-TAGAAACCTCAGCTGGAGGC-3′).

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a-Latroeggtoxin-V were transformed into BL21 (DE3). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a-Latroeggtoxin-V were transformed into BL21 (DE3).

    Article Title: Anti-Breast Cancer Activity of Latroeggtoxin-V Mined from the Transcriptome of Spider Latrodectus tredecimguttatus Eggs
    Article Snippet: After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a- Latroeggtoxin-V were transformed into BL21 (DE3). .. After gel-purification and ligation of the digested products with T4 ligase (Thermo Fisher Scientific, Waltham, MA, USA), the constructed recombinant vectors pET-28a- Latroeggtoxin-V were transformed into BL21 (DE3).

    Lysis:

    Article Title: Chromosome conformation capture of transcriptional interactions between cytochrome c oxidase genes and genes of glutamatergic synaptic transmission in neurons
    Article Snippet: Nuclei were isolated by incubating cells in a lysis buffer (10 mM Tris, pH 8.0, 10 mM NaCl, 0.2% NP-40) on ice with agitation for 30 min. Chromatin was subsequently released by treating isolated nuclei with 0.3% SDS and digested with 400 units of Bgl II (Fermentas, Hanover, MD) overnight. .. Chromatin re-ligation was performed by incubating diluted chromatin with 100 Weiss units of T4 ligase (Fermentas) for 4 h at 16°C.

    Article Title: GA-Binding Protein and p300 Are Essential Components of a Retinoic Acid-Induced Enhanceosome in Myeloid Cells
    Article Snippet: Following centrifugation, the cell pellet was resuspended in 50 ml lysis buffer (10 mM Tris HCl, pH 8.0, 10 mM NaCl, 0.2% NP-40 with protease inhibitors) and incubated on ice for 90 min with mixing. .. Ligation was then performed at 16°C using 30 Weiss units of T4 ligase (Invitrogen) for 4 h. Samples were then treated with proteinase K (final concentration, 100 μg/ml) and incubated overnight at 65°C to reverse cross-links.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 75
    Thermo Fisher t4 dna ligase
    Effects of the distance between a hairpin and the ligation site on the magnitude of ‘terminal hairpin effect’ for the selective formation of single-stranded DNA ring. ( A ) The solution structures of l-DNAs used here. ( B ) Lane 1, L64 2-4,23-4,51-2 without the treatment; lane 2, L64 2-4,23-4,51-2 treated with <t>T4</t> DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of l-DNA; lane 3, L64 3-4,24-4 alone; lane 4, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 5, L64 4-4,25-4 alone; lane 6, L64 4-4,25-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 7, L64 5-4,26-4 alone; lane 8, L64 5–4,26-4 treated with T4 DNA ligase in the presence of 12-nt splint. lane 9, L64 6-4,27-4 alone; lane 10, L64 6-4,27-4 treated with T4 DNA ligase in the presence of 12-nt splint. Reaction conditions are the same as described in Figure 2 .
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 75/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
    Average 75 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2019-12
    75/100 stars
      Buy from Supplier

    Image Search Results


    Effects of the distance between a hairpin and the ligation site on the magnitude of ‘terminal hairpin effect’ for the selective formation of single-stranded DNA ring. ( A ) The solution structures of l-DNAs used here. ( B ) Lane 1, L64 2-4,23-4,51-2 without the treatment; lane 2, L64 2-4,23-4,51-2 treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of l-DNA; lane 3, L64 3-4,24-4 alone; lane 4, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 5, L64 4-4,25-4 alone; lane 6, L64 4-4,25-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 7, L64 5-4,26-4 alone; lane 8, L64 5–4,26-4 treated with T4 DNA ligase in the presence of 12-nt splint. lane 9, L64 6-4,27-4 alone; lane 10, L64 6-4,27-4 treated with T4 DNA ligase in the presence of 12-nt splint. Reaction conditions are the same as described in Figure 2 .

    Journal: Nucleic Acids Research

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    doi: 10.1093/nar/gky769

    Figure Lengend Snippet: Effects of the distance between a hairpin and the ligation site on the magnitude of ‘terminal hairpin effect’ for the selective formation of single-stranded DNA ring. ( A ) The solution structures of l-DNAs used here. ( B ) Lane 1, L64 2-4,23-4,51-2 without the treatment; lane 2, L64 2-4,23-4,51-2 treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of l-DNA; lane 3, L64 3-4,24-4 alone; lane 4, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 5, L64 4-4,25-4 alone; lane 6, L64 4-4,25-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 7, L64 5-4,26-4 alone; lane 8, L64 5–4,26-4 treated with T4 DNA ligase in the presence of 12-nt splint. lane 9, L64 6-4,27-4 alone; lane 10, L64 6-4,27-4 treated with T4 DNA ligase in the presence of 12-nt splint. Reaction conditions are the same as described in Figure 2 .

    Article Snippet: T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques: Ligation

    Effects of the stability of hairpin on the cyclization by T4 DNA ligase. ( A ) The solution conformations of L64 1-4,24-4 , L64 1-6,24-4 , L64 1-7,24-4 and L60 1-7,20-4 , determined by Mfold calculation. ( B ) Lane 1, L64 1-4,24-4 without T4 ligase treatment; lane 2, L64 1-4,24-4 with T4 ligase treatment; lane 3, L64 1-6,24-4 alone; lane 4, L64 1-6,24-4 with T4 ligase treatment; lane 5, L64 1-7,24-4 alone; lane 6, L64 1-7,24-4 with T4 ligase treatment; lane 7, L60 1-7,20-4 alone; lane 8, L60 1-7,20-4 with T4 ligase treatment. The enzymatic conditions are the same as described in Figure 2 .

    Journal: Nucleic Acids Research

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    doi: 10.1093/nar/gky769

    Figure Lengend Snippet: Effects of the stability of hairpin on the cyclization by T4 DNA ligase. ( A ) The solution conformations of L64 1-4,24-4 , L64 1-6,24-4 , L64 1-7,24-4 and L60 1-7,20-4 , determined by Mfold calculation. ( B ) Lane 1, L64 1-4,24-4 without T4 ligase treatment; lane 2, L64 1-4,24-4 with T4 ligase treatment; lane 3, L64 1-6,24-4 alone; lane 4, L64 1-6,24-4 with T4 ligase treatment; lane 5, L64 1-7,24-4 alone; lane 6, L64 1-7,24-4 with T4 ligase treatment; lane 7, L60 1-7,20-4 alone; lane 8, L60 1-7,20-4 with T4 ligase treatment. The enzymatic conditions are the same as described in Figure 2 .

    Article Snippet: T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques:

    Terminal hairpin strategy for T4 DNA ligase-mediated preparation of DNA rings of smaller sizes. ( A ) Solution structures of 74-, 64-, 54-, 44- and 34-nt l-DNAs. ( B ) Gel electrophoresis patterns of the T4 ligase ligation products. The conditions of T4 ligase reactions are the same as described in Figure 2 .

    Journal: Nucleic Acids Research

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    doi: 10.1093/nar/gky769

    Figure Lengend Snippet: Terminal hairpin strategy for T4 DNA ligase-mediated preparation of DNA rings of smaller sizes. ( A ) Solution structures of 74-, 64-, 54-, 44- and 34-nt l-DNAs. ( B ) Gel electrophoresis patterns of the T4 ligase ligation products. The conditions of T4 ligase reactions are the same as described in Figure 2 .

    Article Snippet: T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques: Nucleic Acid Electrophoresis, Ligation

    Dominant cyclization of l-DNA using hairpins as internal promoters. ( A ) The solution structures of L64 3-4,24-4 , L64 16-4,37-4 and L64 3-4 , determined by Mfold calculation under the conditions of [Mg 2+ ] = 10 mM and 25°C. ( B ) Treatments of these l-DNAs with T4 DNA ligase. Lane 1, L64 3-4,24-4 without the T4 ligase treatment; lane 2, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of L64 3-4,24–4 ; lane 4, L64 16-4,37-4 without the treatment; lane 5, L64 16-4,37-4 treated with T4 DNA ligase in the presence of 12-nt splint. Lane 7, L64 3-4 without the treatment; lane 8, L64 3-4 treated with T4 DNA ligase in the presence of 12-nt splint. In lanes 3, 6 and 9, the products in lanes 2, 5 and 8 were further treated with Exonuclease I to remove non-cyclic products. The conditions for the T4 ligase reactions: [l-DNA] 0 = 5 μM, [splint] 0 = 10 μM and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C for 12 h.

    Journal: Nucleic Acids Research

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    doi: 10.1093/nar/gky769

    Figure Lengend Snippet: Dominant cyclization of l-DNA using hairpins as internal promoters. ( A ) The solution structures of L64 3-4,24-4 , L64 16-4,37-4 and L64 3-4 , determined by Mfold calculation under the conditions of [Mg 2+ ] = 10 mM and 25°C. ( B ) Treatments of these l-DNAs with T4 DNA ligase. Lane 1, L64 3-4,24-4 without the T4 ligase treatment; lane 2, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of L64 3-4,24–4 ; lane 4, L64 16-4,37-4 without the treatment; lane 5, L64 16-4,37-4 treated with T4 DNA ligase in the presence of 12-nt splint. Lane 7, L64 3-4 without the treatment; lane 8, L64 3-4 treated with T4 DNA ligase in the presence of 12-nt splint. In lanes 3, 6 and 9, the products in lanes 2, 5 and 8 were further treated with Exonuclease I to remove non-cyclic products. The conditions for the T4 ligase reactions: [l-DNA] 0 = 5 μM, [splint] 0 = 10 μM and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C for 12 h.

    Article Snippet: T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques:

    Highly selective cyclization at unusually high substrate concentrations using terminal hairpin strategy. Lane 1, L64 3-4,24-4 without T4 treatment; lane 2, T4 reaction at [L64 3-4,24-4 ] 0 = 10 μM; lane 3, [L64 3-4,24-4 ] 0 = 20 μM; lane 4, [L64 3-4,24-4 ] 0 = 40 μM; lane 5, [L64 3-4,24-4 ] 0 = 60 μM; lane 6, [L64 3-4,24-4 ] 0 = 100 μM. In lanes 8 - 10, L64 16-4,37-4 having no terminal hairpin is used. Lane 8, L64 16-4,37-4 without T4 treatment; lane 9, [L64 16-4,37-4 ] 0 = 100 μM. Reaction conditions: [l-DNA] 0 /[splint] 0 = 1/2 and 10 U T4 DNA ligase in 1 × T4 DNA ligase buffer at 25°C. In lanes 7 and 10, 0.1× T4 DNA ligase buffer was used in place of 1× T4 buffer, according to ref. ( 30 ) (see text for details).

    Journal: Nucleic Acids Research

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    doi: 10.1093/nar/gky769

    Figure Lengend Snippet: Highly selective cyclization at unusually high substrate concentrations using terminal hairpin strategy. Lane 1, L64 3-4,24-4 without T4 treatment; lane 2, T4 reaction at [L64 3-4,24-4 ] 0 = 10 μM; lane 3, [L64 3-4,24-4 ] 0 = 20 μM; lane 4, [L64 3-4,24-4 ] 0 = 40 μM; lane 5, [L64 3-4,24-4 ] 0 = 60 μM; lane 6, [L64 3-4,24-4 ] 0 = 100 μM. In lanes 8 - 10, L64 16-4,37-4 having no terminal hairpin is used. Lane 8, L64 16-4,37-4 without T4 treatment; lane 9, [L64 16-4,37-4 ] 0 = 100 μM. Reaction conditions: [l-DNA] 0 /[splint] 0 = 1/2 and 10 U T4 DNA ligase in 1 × T4 DNA ligase buffer at 25°C. In lanes 7 and 10, 0.1× T4 DNA ligase buffer was used in place of 1× T4 buffer, according to ref. ( 30 ) (see text for details).

    Article Snippet: T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques:

    Comparison of the reaction conversion and yield of monomeric cyclic ring between substrates with and without the terminal hairpin. ( A ) Time-courses for the T4 ligase-mediated ligation of L64 3-4,24-4 (circles) and L64 16-4,37-4 (rectangles). The total amounts of DNA, consumed in the presence of T4 ligase (by both intramolecular and intermolecular ligation), are plotted as a function of reaction time. In ( B ), the yield of DNA ring is shown as a function of reaction time. Reaction conditions: [l-DNA] 0 = 5 μM, [splint] 0 = 10 μM, and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C.

    Journal: Nucleic Acids Research

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    doi: 10.1093/nar/gky769

    Figure Lengend Snippet: Comparison of the reaction conversion and yield of monomeric cyclic ring between substrates with and without the terminal hairpin. ( A ) Time-courses for the T4 ligase-mediated ligation of L64 3-4,24-4 (circles) and L64 16-4,37-4 (rectangles). The total amounts of DNA, consumed in the presence of T4 ligase (by both intramolecular and intermolecular ligation), are plotted as a function of reaction time. In ( B ), the yield of DNA ring is shown as a function of reaction time. Reaction conditions: [l-DNA] 0 = 5 μM, [splint] 0 = 10 μM, and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C.

    Article Snippet: T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques: Ligation

    Cyclization of l-DNA 66  by T4 DNA ligase. Schematic views of the formation of ( A ) single-stranded DNA ring (c-DNA 66 ) with the assistance of splint-12 nt and ( B ) polymers from multiple l-DNA 66  strands. The single-stranded DNA substrate (l-DNA 66 ) bears a phosphate at the 5′-terminus. ( C ) Effects of [l-DNA 66 ] 0  on the formation of c-DNA 66  and the polymers in 1× T4 ligase buffer (conventional method). The selectivity for the formation of c-DNA 66  is presented below the corresponding band. [l-DNA 66 ] 0 /[splint-12 nt] 0  = 1/2 at 20°C for 12 h. All the DNA substrate was added to the mixture all at once at the beginning of the reaction.

    Journal: Nucleic Acids Research

    Article Title: Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration

    doi: 10.1093/nar/gkx553

    Figure Lengend Snippet: Cyclization of l-DNA 66 by T4 DNA ligase. Schematic views of the formation of ( A ) single-stranded DNA ring (c-DNA 66 ) with the assistance of splint-12 nt and ( B ) polymers from multiple l-DNA 66 strands. The single-stranded DNA substrate (l-DNA 66 ) bears a phosphate at the 5′-terminus. ( C ) Effects of [l-DNA 66 ] 0 on the formation of c-DNA 66 and the polymers in 1× T4 ligase buffer (conventional method). The selectivity for the formation of c-DNA 66 is presented below the corresponding band. [l-DNA 66 ] 0 /[splint-12 nt] 0 = 1/2 at 20°C for 12 h. All the DNA substrate was added to the mixture all at once at the beginning of the reaction.

    Article Snippet: T4 DNA ligase was purchased from Thermo Scientific (Pittsburgh, PA, USA), together with the 10 × T4 ligase buffer.

    Techniques:

    Effects of ( A ) [Mg 2+ ] 0  and ( B ) [ATP] 0  on the cyclization of l-DNA 66  by T4 DNA ligase. [l-DNA 66 ] 0  = 1 μM, [splint-12 nt] 0  = 2 μM, 5 U T4 DNA ligase at 20°C and 12 h. [ATP] 0  = 25 μM, [DTT] = 0.5 mM, and [Tris–HCl] = 2 mM in ( A ), whereas [MgCl 2 ] = 0.5 mM, [DTT] = 0.5 mM, and [Tris–HCl] = 2 mM in ( B ).

    Journal: Nucleic Acids Research

    Article Title: Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration

    doi: 10.1093/nar/gkx553

    Figure Lengend Snippet: Effects of ( A ) [Mg 2+ ] 0 and ( B ) [ATP] 0 on the cyclization of l-DNA 66 by T4 DNA ligase. [l-DNA 66 ] 0 = 1 μM, [splint-12 nt] 0 = 2 μM, 5 U T4 DNA ligase at 20°C and 12 h. [ATP] 0 = 25 μM, [DTT] = 0.5 mM, and [Tris–HCl] = 2 mM in ( A ), whereas [MgCl 2 ] = 0.5 mM, [DTT] = 0.5 mM, and [Tris–HCl] = 2 mM in ( B ).

    Article Snippet: T4 DNA ligase was purchased from Thermo Scientific (Pittsburgh, PA, USA), together with the 10 × T4 ligase buffer.

    Techniques:

    Effects of the concentration of T4 ligase buffer on the efficiencies of cyclization of l-DNA 66  and its polymerization. All the DNA substrate was added to the mixture all at once at the beginning of the reaction. The selectivity for the formation of c-DNA 66  is presented below the corresponding band. The reaction conditions: [l-DNA 66 ] 0  = 1 μM; [splint-12 nt] 0  = 2 μM; 5 U T4 DNA ligase at 20°C and 12 h. Note that 1 × T4 ligase buffer contains 10 mM MgCl 2 , 500 μM ATP, 10 mM DTT and 40 mM Tris-HCl.

    Journal: Nucleic Acids Research

    Article Title: Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration

    doi: 10.1093/nar/gkx553

    Figure Lengend Snippet: Effects of the concentration of T4 ligase buffer on the efficiencies of cyclization of l-DNA 66 and its polymerization. All the DNA substrate was added to the mixture all at once at the beginning of the reaction. The selectivity for the formation of c-DNA 66 is presented below the corresponding band. The reaction conditions: [l-DNA 66 ] 0 = 1 μM; [splint-12 nt] 0 = 2 μM; 5 U T4 DNA ligase at 20°C and 12 h. Note that 1 × T4 ligase buffer contains 10 mM MgCl 2 , 500 μM ATP, 10 mM DTT and 40 mM Tris-HCl.

    Article Snippet: T4 DNA ligase was purchased from Thermo Scientific (Pittsburgh, PA, USA), together with the 10 × T4 ligase buffer.

    Techniques: Concentration Assay

    Effects of the length of splints on the efficiencies of cyclization of l-DNA 66  and its polymerization under conventional conditions. All the DNA substrate was added to the mixture all at once at the beginning of the reaction. Each of the splints is complementary to equal number of nucleotides in the 5′- and 3′-ends of l-DNA 66 , respectively (the binding mode of splint-12 nt is presented in Figure   1A ). The reaction conditions: [l-DNA 66 ] 0  = 5 μM; [splint] 0  = 10 μM; 20 U T4 DNA ligase in 1 × T4 ligase buffer at 20°C and 12 h. The sequences of splints were listed in   Supplementary Table S1 .

    Journal: Nucleic Acids Research

    Article Title: Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration

    doi: 10.1093/nar/gkx553

    Figure Lengend Snippet: Effects of the length of splints on the efficiencies of cyclization of l-DNA 66 and its polymerization under conventional conditions. All the DNA substrate was added to the mixture all at once at the beginning of the reaction. Each of the splints is complementary to equal number of nucleotides in the 5′- and 3′-ends of l-DNA 66 , respectively (the binding mode of splint-12 nt is presented in Figure 1A ). The reaction conditions: [l-DNA 66 ] 0 = 5 μM; [splint] 0 = 10 μM; 20 U T4 DNA ligase in 1 × T4 ligase buffer at 20°C and 12 h. The sequences of splints were listed in Supplementary Table S1 .

    Article Snippet: T4 DNA ligase was purchased from Thermo Scientific (Pittsburgh, PA, USA), together with the 10 × T4 ligase buffer.

    Techniques: Binding Assay

    Single-stranded DNA ligation with  T4  DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either  T4  DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok  et al . (  19 ) is shown for comparison. M: Single-stranded DNA size marker.

    Journal: Nucleic Acids Research

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    doi: 10.1093/nar/gkx033

    Figure Lengend Snippet: Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.

    Article Snippet: Ligation was performed in 80 μl reactions containing 1 × T4 RNA ligase buffer, 20% PEG-8000, 0.5 mM ATP, 10/20 pmol of adapter splinter mix CL78/TL38, 1, 2 or 4 pmol acceptor oligonucleotide and 30 U T4 DNA ligase (ThermoFisher Scientific).

    Techniques: DNA Ligation, Ligation, Staining, Marker

    Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with  T4  DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of  Escherichia coli  DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by  T4  DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, (  4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of  Bst  DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using  T4  DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, (  23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using  T4  DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of  E. coli  DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with  T4  DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.

    Journal: Nucleic Acids Research

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    doi: 10.1093/nar/gkx033

    Figure Lengend Snippet: Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.

    Article Snippet: Ligation was performed in 80 μl reactions containing 1 × T4 RNA ligase buffer, 20% PEG-8000, 0.5 mM ATP, 10/20 pmol of adapter splinter mix CL78/TL38, 1, 2 or 4 pmol acceptor oligonucleotide and 30 U T4 DNA ligase (ThermoFisher Scientific).

    Techniques: Hybridization, Ligation, Modification, DNA Ligation, DNA Purification, Mutagenesis, Purification

    Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and  T4  DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.

    Journal: Nucleic Acids Research

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    doi: 10.1093/nar/gkx033

    Figure Lengend Snippet: Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.

    Article Snippet: Ligation was performed in 80 μl reactions containing 1 × T4 RNA ligase buffer, 20% PEG-8000, 0.5 mM ATP, 10/20 pmol of adapter splinter mix CL78/TL38, 1, 2 or 4 pmol acceptor oligonucleotide and 30 U T4 DNA ligase (ThermoFisher Scientific).

    Techniques: Ligation, Sequencing, Ancient DNA Assay, Gas Chromatography