Journal: PLoS ONE

Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

doi: 10.1371/journal.pone.0020556

Figure Lengend Snippet: Test of QC cloning using Klenow DNA polymerase. (A) Test of Klenow exonuclease activity determined using the same assay used for T4 DNA polymerase. (B) To test QC cloning using Klenow DNA polymerase, the PCR product T019 GC3F was cloned into pICH31477 (23 nucleotide catching sequence) and pICH31480 (52 nucleotide catching sequence). Incubation was performed at 37°C for 0, 30, 60, 90, and 120 minutes. ( C ) Eight randomly chosen clones from 120 min time points were analyzed by colony PCR using vector primers. The size of the expected full-length fragment is indicated by an arrow.

Article Snippet: To perform the QC cloning 2 µl PCR product, 1 µl Bpi I-digested vector, 2 µl 10x T4 DNA polymerase buffer, 0.5 µl T4 DNA polymerase (New England Biolabs, Ipswich MA, USA; 3 units/ µl) and 14.5 µl water were mixed and incubated for 5 minutes at room temperature.

Techniques: Clone Assay, Activity Assay, Polymerase Chain Reaction, Sequencing, Incubation, Plasmid Preparation

Journal: PLoS ONE

Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

doi: 10.1371/journal.pone.0020556

Figure Lengend Snippet: Strategy for amplification and QC cloning of immunoglobulin fragments. ( A ) Amplification of immunoglobulin fragments from non-Hodgkin lymphoma samples. Total RNA extracted from biopsy samples (1) is reverse-transcribed into first strand cDNA using an oligo dT primer (2). The cDNA is column-purified to remove remaining dNTPs, and G-tailed using terminal transferase and dGTP (3). (4) The G-tailed cDNA is used as a template for PCR amplification using a G-tail adaptor primer (bap2 pc) and an immunoglobulin constant region-specific primer (gsp). The PCR product is column-purified to remove the remaining dNTPs (5). ( B ) Preparation of vector for QC cloning. The cloning vector is linearized using the enzyme Pst I. ( C ) The column-purified PCR product and the linearized vector are mixed and treated with T4 DNA polymerase to generate single-stranded ends that are complementary between the vector and insert (7). The mixture is directly transformed into chemo-competent E. coli DH10B cells where the annealed ends of the vector and insert complex are repaired and ligated (8). (9) After cloning, the plasmid is purified and the insert sequenced using a vector specific primer (seqpr).

Article Snippet: To perform the QC cloning 2 µl PCR product, 1 µl Bpi I-digested vector, 2 µl 10x T4 DNA polymerase buffer, 0.5 µl T4 DNA polymerase (New England Biolabs, Ipswich MA, USA; 3 units/ µl) and 14.5 µl water were mixed and incubated for 5 minutes at room temperature.

Techniques: Amplification, Clone Assay, Purification, Polymerase Chain Reaction, Plasmid Preparation, Transformation Assay

Journal: PLoS ONE

Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

doi: 10.1371/journal.pone.0020556

Figure Lengend Snippet: Test of QC cloning performed with or without heat inactivation. ( A ) PCR product amplified from G-tailed cDNA prepared from biopsy sample T019 using primers bap2 pc and GC3F. ( B ) Structure of the vector and of the PCR product. ( C , D ) The PCR product was cloned into pICH31480 using T4 DNA polymerase treatment for 5 minutes at 25°C (A, adaptor; U, unknown sequence; K, known sequence; CS, catching sequence), followed by heat inactivation 20 min at 75°C ( C ) or incubation at 4°C ( D ). Eight randomly chosen clones were analyzed by colony PCR using vector primers. The products amplified by colony PCR were separated on a 1% agarose gel supplemented with ethidium bromide and visualized under UV light. The expected insert size is indicated by an arrow.

Article Snippet: To perform the QC cloning 2 µl PCR product, 1 µl Bpi I-digested vector, 2 µl 10x T4 DNA polymerase buffer, 0.5 µl T4 DNA polymerase (New England Biolabs, Ipswich MA, USA; 3 units/ µl) and 14.5 µl water were mixed and incubated for 5 minutes at room temperature.

Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing, Incubation, Agarose Gel Electrophoresis

Journal: PLoS ONE

Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

doi: 10.1371/journal.pone.0020556

Figure Lengend Snippet: Quantification of T4 DNA polymerase exonuclease activity. Sac II/ Nde I-digested plasmid DNA (3 fragments, lane C) was treated with T4 DNA polymerase for 10 minutes at 25°C, 20°C, 15°C and 10°C. The T4 DNA polymerase was then inactivated by incubation at 80°C for 5 min. The single-stranded ends generated by the 3′ to 5′ exonuclease activity T4 DNA polymerase were removed by using Mung Bean nuclease. The size of the resulting fragments was analyzed by agarose gel electrophoresis. As a control for the heat inactivation of T4 DNA polymerase, digested plasmid DNA was inactivated at 80°C for 5 minutes immediately after addition of T4 DNA polymerase (lane H).

Article Snippet: To perform the QC cloning 2 µl PCR product, 1 µl Bpi I-digested vector, 2 µl 10x T4 DNA polymerase buffer, 0.5 µl T4 DNA polymerase (New England Biolabs, Ipswich MA, USA; 3 units/ µl) and 14.5 µl water were mixed and incubated for 5 minutes at room temperature.

Techniques: Activity Assay, Plasmid Preparation, Incubation, Generated, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: An efficient method to assemble linear DNA templates for in vitro screening and selection systems

doi: 10.1093/nar/gkp589

Figure Lengend Snippet: Efficiency of template assembly by uracil excision–ligation and the purifying PCR for DNA templates prepared with Taq DNA polymerase. Precipitation with PEG–MgCl 2 is necessary and sufficient for the efficient assembly. L1, hyperladder I in kilobase pairs; L2, substrate AGT amplified with Taq , precipitated with PEG–MgCl 2 and blunted with T4 DNA polymerase; L3, assembly of L2 with its UTRs + Avi-tag; L4, purifying PCR of L3 with 50 ng DNA per 100 μl PCR; L5, purifying PCR of L3 with 250 ng DNA per 100 μl PCR; L6, purifying PCR of L3 with 500 ng DNA per 100 μl PCR; L7, hyperladder I in kb; L8, substrate AGT amplified with Taq and blunted with T4 DNA polymerase; L9, assembly of L8 with its UTRs + Avi-tag; L10, purifying PCR of L9 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.

Article Snippet: Where applicable, assembly substrates were blunted with T4 DNA polymerase according to the manufacturer's instructions (NEB).

Techniques: Ligation, Polymerase Chain Reaction, Amplification

Journal: Nucleic Acids Research

Article Title: An efficient method to assemble linear DNA templates for in vitro screening and selection systems

doi: 10.1093/nar/gkp589

Figure Lengend Snippet: ( A ) The enrichment of Avi-AGT DNA in model affinity selections relative to a non-binding, control template coding for AGT-FRB was > 250-fold. L1, hyperladder I in kilobase pairs; L2, PCR amplification of the supernatant; L3, PCR amplification of the bead fraction. ( B ) Assembly of recovered DNA fragments; L4, hyperladder I in kilobase pairs; L5, substrate AGT recovered from the bead fraction with PfuTurbo C x , precipitated with PEG–MgCl 2 and blunted with T4 DNA polymerase; L6, assembly of L5 with its UTRs + Avi-tag. L7: purifying PCR of L6 with 50 ng DNA per 100 μl PCR; L8, hyperladder I in kilobase pairs; L9, substrate AGT recovered from the bead fraction with PfuTurbo C x and blunted with T4 DNA polymerase; L10, assembly of L9 with its UTRs + Avi-tag; L11, purifying PCR of L10 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.

Article Snippet: Where applicable, assembly substrates were blunted with T4 DNA polymerase according to the manufacturer's instructions (NEB).

Techniques: Binding Assay, Polymerase Chain Reaction, Amplification

Journal: Nucleic Acids Research

Article Title: An efficient method to assemble linear DNA templates for in vitro screening and selection systems

doi: 10.1093/nar/gkp589

Figure Lengend Snippet: ( A ) Assembly Scheme. (i) GOI or a derivative library is amplified with primers that specifically incorporate uracil nucleotides close to both 5′-ends. (ii) Assembly of the GOI with its 5′- and 3′-untranslated regions including any constant protein-coding regions based on a coupled uracil excision–ligation strategy. (iii) Pure templates are obtained following a short-purifying PCR, which effectively ‘removes’ excess substrates and partially assembled intermediates. ( B ) Mechanism of coupled uracil excision–ligation: first, USER enzyme catalyses the excision of uracil from DNA, thereby leaving a single base pair gap and a 3′-extension provided the 5′-portion can dissociate. Complementary overlapping 3′-extensions then direct the assembly of DNA fragments which are covalently sealed by T4 DNA ligase.

Article Snippet: Where applicable, assembly substrates were blunted with T4 DNA polymerase according to the manufacturer's instructions (NEB).

Techniques: Amplification, Ligation, Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: An efficient method to assemble linear DNA templates for in vitro screening and selection systems

doi: 10.1093/nar/gkp589

Figure Lengend Snippet: Assembly of an epPCR library. L1, hyperladder I in kilobase pairs;. L2, epPCR library prepared with the Genemorph II kit; L3, epPCR library of L2 re-amplified with Taq DNA polymerase and uracil-containing primers; L4, assembly of L3 with its UTRs+Avi-tag after it has been precipitated with PEG–MgCl 2 and blunted with T4 DNA polymerase; L5, purifying PCR of L4 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.

Article Snippet: Where applicable, assembly substrates were blunted with T4 DNA polymerase according to the manufacturer's instructions (NEB).

Techniques: Amplification, Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

doi: 10.1038/s41598-018-34079-2

Figure Lengend Snippet: CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.

Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

Techniques: Labeling, Gas Chromatography, Incubation, Negative Control

Journal: Scientific Reports

Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

doi: 10.1038/s41598-018-34079-2

Figure Lengend Snippet: Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 -benzylguanine (BG) moieties that specifically react with active site cysteine residues of SNAP-tag proteins, forming a stable covalent thioether bond 15 , 16 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

Techniques: Conjugation Assay, Magnetic Beads, Sequencing, Modification, Amplification, Polymerase Chain Reaction, Purification, Selection

Journal: Scientific Reports

Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

doi: 10.1038/s41598-018-34079-2

Figure Lengend Snippet: A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c ) Processing AT-rich DNA by Taq DNA pol at elevated temperatures. During high temperature incubation, for example, at 65 °C or 70 °C, the ends of AT-rich DNA fragments melt into transient or predominant single-stranded structures. Taq DNA pol (red) can act on these DNA substrates by its polymerization and 5′ nuclease activities as previously described 34 , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.

Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

Techniques: Gas Chromatography, Sequencing, Amplification, Next-Generation Sequencing, Activity Assay, Incubation, Activated Clotting Time Assay

Journal: PLoS ONE

Article Title: Trypanosoma brucei TIF2 and TRF Suppress VSG Switching Using Overlapping and Independent Mechanisms

doi: 10.1371/journal.pone.0156746

Figure Lengend Snippet: Tb TRF depletion does not affect the amount of DSBs inside VSG ESs. (A) Principle of LMPCR assay. After DSBs (represented by a bolt) form, an adapter is ligated with the genomic DNA at the break sites if they have blunt ends. Treating genomic DNA with T4 DNA polymerase converts staggered broken ends into blunt ends. The ligated products are then amplified by PCR using a locus-specific forward primer and the adapter-specific reverse primer. The PCR amplified products are subsequently detected by locus-specific probes in Southern analysis. (B C) LMPCR analyses were performed in Tb TRF RNAi cells. The LMPCR products were hybridized with VSG2 (B) and VSG21 (C). In panels B C, the Ethidium Bromide (EtBr)-stained LMPCR products are shown at the top, the Southern blot result is shown in the middle, and the PCR products using primers specific to the TbRAP1 gene (as a loading control) are shown at the bottom. The amounts of input genomic DNA, either treated (+) or not treated (−) with T4 DNA polymerase, were marked on top of each lane. (D) Quantification of the change in the amounts of LMPCR products (with T4 DNA pol treatment using 54 ng input gDNA) from three independent experiments. Average values are shown. Error bars represent standard deviations.

Article Snippet: Briefly, in each ligation reaction, 2 μg of genomic DNA was either treated or not treated with 2 μl of T4 DNA Polymerase (3000 U/ml, New England BioLabs) in the presence of 200 μM dNTP and then ligated with 10 μl annealed adaptor.

Techniques: Amplification, Polymerase Chain Reaction, Staining, Southern Blot

Journal: PLoS ONE

Article Title: Trypanosoma brucei TIF2 and TRF Suppress VSG Switching Using Overlapping and Independent Mechanisms

doi: 10.1371/journal.pone.0156746

Figure Lengend Snippet: Expression of ectopic F2H- Tb TRF does not suppress the phenotype of increased subtelomeric DSB numbers in Tb TIF2 RNAi cells. LMPCR analyses were performed in S/TIF2i+F2H- Tb TRF cells. LMPCR products were hybridized with VSG pseudogene ψ ES1 (A) and VSG21 (B). Ethidium Bromide (EtBr)-stained LMPCR products are shown at the top, the Southern blot result is shown in the middle, and the PCR products using primers specific to the TbRAP1 gene (as a loading control) are shown at the bottom. (C) Quantification of the change in the amounts of LMPCR products (with T4 DNA pol treatment using 54 ng input gDNA) from three independent experiments. Average values are shown. Error bars represent standard deviations. Numbers represent P values of unpaired t-tests between pairs of data groups as indicated.

Article Snippet: Briefly, in each ligation reaction, 2 μg of genomic DNA was either treated or not treated with 2 μl of T4 DNA Polymerase (3000 U/ml, New England BioLabs) in the presence of 200 μM dNTP and then ligated with 10 μl annealed adaptor.

Techniques: Expressing, Staining, Southern Blot, Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL

doi: 10.1093/nar/gky708

Figure Lengend Snippet: FEN1 completes primer removal and L-strand maturation. ( A ) Schematic of template used in panel B. The possible products are illustrated (b–d). ( B ) Coupled nuclease gap-filling ligation assay was performed as in Figure 3 but in the presence of the indicated nucleases. MGME1 (250 fmol) was added in lanes 4 and 9; FEN1 (35 fmol) was added in lanes 5 and 10. The samples in lanes 6–10 contained 300 fmol DNA ligase III. Conversion of the nicked product (c) to a ligated 80 nt product (d) was stimulated when FEN1 was added together with RNase H1. ( C ) Schematic of template used in panel D. The downstream chimeric oligonucleotide was labelled at the 3′-end. The possible products are illustrated (b–d). ( D ) Coupled nuclease gap-filling ligation assay was performed in the presence of RNase H1 alone or together with FEN1 (35 fmol) as indicated. Lanes 2–4 contained only RNase H1 with or without FEN1 to monitor nuclease activity. RNase H1 cleaved the downstream oligonucleotide leaving 1–3 unprocessed ribonucleotides. FEN1 cleaved the remaining ribonucleotides, resulting in a shorter product. POLγ and DNA ligase (ligase III or T4 DNA ligase) were added in lanes 5–10. Ligase III showed ligation only when both RNase H1 and FEN1 were added. However, T4 DNA ligase could ligate without FEN1. Lanes 12–14 are ligation controls using a 3′-end labelled DNA-only oligonucleotide with a 5′-end phosphate. Note that the short band (

Article Snippet: For 3′-end labelling, a 51 nt long 26RNA:25DNA oligonucleotide was radiolabelled to generate a 52 nt long 26RNA:26DNA oligonucleotide by one of two methods: 3′-end labelling using Klenow fragment fill-in and [α-32 P] dCTP; or, 3′-end labelling using terminal transferase (NEB) and [α-32 P] dCTP, followed by 3′ overhang removal using T4 DNA polymerase (NEB).

Techniques: Ligation, Activity Assay

Journal: Nucleic Acids Research

Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL

doi: 10.1093/nar/gky708

Figure Lengend Snippet: RNase H1 processing coupled to POLγ dependent DNA synthesis does not produce ligatable nicks. ( A ) Schematic of the coupled nuclease gap-filling ligation assay performed on a gapped OriL substrate (a). The upstream oligonucleotide was radioactively labelled at the 5′-end. The possible products are illustrated (b–d). (–) Coupled nuclease gap-filling ligation assay as shown in A. POLγ filled the gap (lane 2, marked b) and had limited strand displacement activity (SD). Note though that POLγ completely displaces the downstream oligonucleotide in a small fraction of templates (80 nt band, lanes 3–6,), RNase H1 cleaved the RNA in the substrate (lane 3), enabling further gap-filling (marked c). Only very low levels of ligated products were formed in the presence of 80–320 fmol DNA ligase III (lanes 4–6). A prominent 80 nt ligated product was formed with T4 DNA ligase (lane 7, marked d). The letters a-d correspond to the illustrations in panel A. ( C ) Ligation assay on a nicked substrate containing RNA tracts of varying length downstream of the nick in the presence of 80–320 fmol DNA ligase III. DNA ligase III discriminates against nicked substrates that contain increasing stretches of ribonucleotides. Two, but not five or more ribonucleotides, can be ligated. ( D ) As in C, except performed with T4 ligase (1–8 U). T4 ligase can ligate five but not 10 ribonucleotides. ( E ) T4 ligase-mediated ligation is abolished in the presence of the mutant RNase H1 proteins. The letters a-d correspond to the illustrations in panel A.

Article Snippet: For 3′-end labelling, a 51 nt long 26RNA:25DNA oligonucleotide was radiolabelled to generate a 52 nt long 26RNA:26DNA oligonucleotide by one of two methods: 3′-end labelling using Klenow fragment fill-in and [α-32 P] dCTP; or, 3′-end labelling using terminal transferase (NEB) and [α-32 P] dCTP, followed by 3′ overhang removal using T4 DNA polymerase (NEB).

Techniques: DNA Synthesis, Ligation, Activity Assay, Mutagenesis

Journal: PLoS ONE

Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

doi: 10.1371/journal.pone.0018900

Figure Lengend Snippet: Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.

Article Snippet: To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

Techniques: Generated, Expressing, Plasmid Preparation, Sequencing, Marker

Journal: PLoS ONE

Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

doi: 10.1371/journal.pone.0018900

Figure Lengend Snippet: Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

Article Snippet: To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

Techniques: Ligation, Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Transformation Assay

Journal: PLoS ONE

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

doi: 10.1371/journal.pone.0111538

Figure Lengend Snippet: Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).

Article Snippet: Both of these enzymes are then heat-inactivated in the same tube, followed by addition of dTTP and T4 DNA polymerase without the need for adding any buffer, as T4 DNA polymerase is compatible with most buffers used for PCR.

Techniques: Clone Assay, Plasmid Preparation, Sequencing, Amplification, Polymerase Chain Reaction, Ligation

Journal: Nucleic Acids Research

Article Title: DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells

doi: 10.1093/nar/gkt804

Figure Lengend Snippet: Human DHX9 recognizes DNA secondary structures in plasmids. ( A ), representative agarose gel of CC pMEXr (lanes 1–5) and pCEX (lanes 6–10) incubated with 30 nM purified recombinant human DHX9 protein (lanes 3–5 and 8–10) in the presence of 5 mM ATP (lanes 3 and 4, and 8 and 9) or 5 mM AMP-PNP (lanes 5 and 10) followed by MBN cleavage. Lane M, 1 kb DNA marker. ( B ), plot of net percentage of OC and linear ( L ) DNA released from total DNA ( OC + L + CC ) ( CC = closed circular DNA) at the end of the reaction from two experiments, as described in Panel (A). The net percentage was defined as F = f s – f c , were f s was the %[( OC + L )/( OC + L + CC )] for each sample, and f c was the average %[( OC + L )/( OC + L + CC )] for the two samples without MBN (i.e. lanes 1, 3 and 6, 8, respectively). ( C ) , schematic of the diagnostic restriction sites (AhdI and EcoO109I) used to map MBN-specific cleavage in pMEXr. Grey arrows, lengths of restriction fragments released from pCEX and pMEXr when cleaved by AhdI, EcoO109I and EcoRI, which are located several bp from the cloned inserts; C/H, position of the control (C) and H-DNA-forming (H) inserts in pCEX and pMEXr, respectively. ( D ), MBN cleavage mapping. PhosphorImager scan of an agarose gel after electrophoresis of pCEX (lanes 1–4) and pMEXr (lanes 5–8) pre-incubated with 30 nM DHX9 (lanes 2 and 3, and 6 and 7) in the presence of 5 mM ATP, treated with 40 units MBN (lanes 3 and 4, and 7 and 8), end-labeled with T4 DNA polymerase and cleaved with Eco0109I and AhdI. Lane E, control lane containing pMEXr cleaved with EcoRI, labeled with T4 DNA polymerase, and then cleaved with AhdI and EcoO109I; E* , ethidium bromide staining of lane E.

Article Snippet: DNA was labeled with 10 units T4 DNA polymerase (New England Biolabs) in a 18 µl of reaction volume containing the manufacturer recommended buffer at 12°C for 7 min.

Techniques: Agarose Gel Electrophoresis, Incubation, Purification, Recombinant, Marker, Diagnostic Assay, Clone Assay, Electrophoresis, Labeling, Staining

Journal: PLoS ONE

Article Title: Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection

doi: 10.1371/journal.pone.0191315

Figure Lengend Snippet: Diagrammatic representation of T7 promoter-lac operator-based pVMExp14367 expression vector to obtain recombinant proteins with N-terminal H10-TEV and C-terminal BAP tag. Only relevant genes and restriction sites are shown. The maps are not to scale. T7 lac , T7 promoter-lac operator; RBS, ribosome-binding site; H10, deca-histidine tag; TEV, Tobacco Etch Virus protease cleavage site; S, glycine-serine rich spacers; SacR-SacB, 2.0 kbp SacR-SacB gene cassette flanked by BsaI sites; BAP, Biotin Acceptor Peptide; T7Tn, T7 transcription terminator; f ori, origin of replication of filamentous phage; Amp r , beta-lactamase gene; ColE1 ori, origin of replication. The amino acids encoded are shown in single letter code (bold) above the nucleotide sequence (A-D). (A-C) The sequence of the important components of vector including 2 BsaI cloning sites. (D) Sequence flanking the gene of interest (GOI) after PCR amplification. (E) GOI carrying 5’- 4 base overhangs generated after treatment with T4 DNA polymerase in the presence of dTTP.

Article Snippet: Restriction enzymes, T4 DNA ligase, and T4 DNA polymerase were obtained from NEB, Ipswich, MA, USA.

Techniques: Expressing, Plasmid Preparation, Recombinant, Binding Assay, Sequencing, Clone Assay, Polymerase Chain Reaction, Amplification, Generated

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: For fill-in with T4 DNA polymerase a 50 μl reaction mix was prepared containing 1× T4 DNA polymerase buffer (ThermoFisher Scientific), 0.05% Tween-20, 100 μM each dNTP, 100 pmol primer CL130 and 2 μl 5 U/μl T4 DNA polymerase (ThermoFisher Scientific).

Techniques: DNA Ligation, Ligation, Staining, 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: 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: For fill-in with T4 DNA polymerase a 50 μl reaction mix was prepared containing 1× T4 DNA polymerase buffer (ThermoFisher Scientific), 0.05% Tween-20, 100 μM each dNTP, 100 pmol primer CL130 and 2 μl 5 U/μl T4 DNA polymerase (ThermoFisher Scientific).

Techniques: Hybridization, Ligation, Modification, DNA Ligation, DNA Purification, Mutagenesis, 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: 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: For fill-in with T4 DNA polymerase a 50 μl reaction mix was prepared containing 1× T4 DNA polymerase buffer (ThermoFisher Scientific), 0.05% Tween-20, 100 μM each dNTP, 100 pmol primer CL130 and 2 μl 5 U/μl T4 DNA polymerase (ThermoFisher Scientific).

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

Journal: Scientific Reports

Article Title: A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses

doi: 10.1038/srep06438

Figure Lengend Snippet: Absolute quantification of Tomato yellow leaf curl Sardinia virus (TYLCSV) virion-sense (VS) and complementary-sense (CS) ssDNA molecules, in the presence of an equimolar amount of dsDNA molecules of the same virus. Circular ssDNA molecules bearing the TYLCSV VS strand (10 6 copies) were mixed with the same quantity of circular ssDNA bearing the TYLCSV CS strand and with 10 6 molecules of phagemid dsDNA carrying the TYLCSV genome. The mix was denatured and used as template for T4 DNA polymerase primer extension with the primers described in Fig. 1 , followed by qPCR with the indicated primer combinations for quantification of VS strands (grey bar), CS strands (white bar) and total viral DNA (VS and CS strands) (black bar). Data represent the average of three technical qPCR replicates.

Article Snippet: T4 DNA polymerase (TAKARA, Shiga, Japan) was used for the extension reaction.

Techniques: Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses

doi: 10.1038/srep06438

Figure Lengend Snippet: Schematic representation of a two-step quantitative PCR (qPCR) procedure for the quantification of virion-sense (VS) and complementary-sense (CS) DNA molecules. (A) Amplification of the VS strand using the OCS-TAG primer for T4 DNA polymerase extension and subsequent qPCR amplification with OVS and TAG primers. (B) Amplification of the CS strand using the OVS-TAG primer for T4 DNA polymerase extension followed by qPCR amplification with OCS and TAG primers. (C) qPCR to quantify both VS and CS strands using OVS and OCS primers. Primers used for T4 polymerase extension are removed prior to performing qPCR.

Article Snippet: T4 DNA polymerase (TAKARA, Shiga, Japan) was used for the extension reaction.

Techniques: Real-time Polymerase Chain Reaction, Amplification