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: PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 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: PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 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: 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: PLoS ONE

Article Title: A Rapid Cloning Method Employing Orthogonal End Protection

doi: 10.1371/journal.pone.0037617

Figure Lengend 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.

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.

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

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

Journal: Molecular and Cellular Biology

Article Title: Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

doi:

Figure Lengend Snippet: J κ 1 and J H 2 coding ends have 3′ overhangs. (A and B) DNA purified in agarose plugs from newborn-mouse thymocytes (A) or 103 bcl2/4 cells cultured at 33 or 39°C (B) (shift − or +) was ligated to the BW linker without pretreatment (lanes none) or after treatment with either T4 DNA polymerase (lanes T4 Pol) or mung bean nuclease (lanes MB-N). Ligated plugs were analyzed by PCR for J H 2 (A) or J κ 1 (B) coding-end (ce) breaks (arrows). The lanes labeled 63-12 were control LM-PCR assays with 63-12 cell DNA. Lanes 1 and 2 in panel A represent independent thymocyte DNA samples. (C) Amplified products from lanes 1, 3, and 4 in panel A and lanes 2, 4, and 6 in panel B were gel purified, reamplified for five cycles with a 32 P-labeled specific oligonucleotide, and analyzed by denaturing polyacrylamide gel electrophoresis. In each case, the arrow indicates the predominant broken-ended molecule (+9 for J H 2 and +4 for J κ 1) and the tick marks indicate 1-nt intervals (determined by the electrophoresis of a DNA sequencing reaction mixture on the same gel). Lanes: N, no pretreatment; T, T4 DNA polymerase pretreatment; M, mung bean nuclease pretreatment.

Article Snippet: Some plug DNA samples were subjected to T4 DNA polymerase treatment by incubating 40 μl of plug in an 80-μl reaction mixture with manufacturer’s buffer (Life Technologies), 5 U of T4 DNA polymerase, and 100 μM deoxynucleoside triphosphates at 37°C for 1 h. Other plugs were treated with various amounts of mung bean nuclease (BRL) under conditions previously reported by Zhu and Roth ( ).

Techniques: Purification, Cell Culture, Polymerase Chain Reaction, Labeling, Amplification, Polyacrylamide Gel Electrophoresis, Electrophoresis, DNA Sequencing

Journal: Molecular and Cellular Biology

Article Title: Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

doi:

Figure Lengend Snippet: V(D)J recombination reaction pathway and LM-PCR assay for reaction intermediates. (A) Diagram of the reactants (top), broken DNA intermediates (middle), and products (bottom) of V(D)J recombination. V and J gene segments, with their associated RSSs (heptamer [H] and nonamer [N]) are recognized and cleaved by the recombinase at the RSS–coding-segment junction (arrow), generating coding-end and signal-end fragments. These ends are joined to form a coding joint and a signal joint. (B) LM-PCR assay for broken-ended recombination reaction intermediates. The BW linker is ligated to available ends in total genomic DNA by using T4 DNA ligase. The sites of linker ligation are revealed by a set of nested PCR assays with a linker primer (BW-1) and locus-specific primers (open arrows labeled 1, 2, 4, and 5). Blots of PCR products were probed with internal oligonucleotides (solid lines labeled 3 and 6).

Article Snippet: Some plug DNA samples were subjected to T4 DNA polymerase treatment by incubating 40 μl of plug in an 80-μl reaction mixture with manufacturer’s buffer (Life Technologies), 5 U of T4 DNA polymerase, and 100 μM deoxynucleoside triphosphates at 37°C for 1 h. Other plugs were treated with various amounts of mung bean nuclease (BRL) under conditions previously reported by Zhu and Roth ( ).

Techniques: Polymerase Chain Reaction, Ligation, Nested PCR, Labeling

Journal: Molecular and Cellular Biology

Article Title: Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

doi:

Figure Lengend Snippet: T4 DNA polymerase treatment enhances LM-PCR detection of broken coding ends. (A to C) DNA samples prepared by the agarose plug method from uninduced (33°C [33 degr]) and induced (39°C [39 degr]) 103 bcl2/4 cells (A and B) and from newborn thymus (C) were analyzed by LM-PCR for broken J κ 1 coding (A), J κ 1 and J κ 2 signal (B), and J H 2 coding and signal (C) ends without (lanes −) or with (lanes +) T4 DNA polymerase (T4 pol) pretreatment. Controls included identically prepared and treated 63-12 (RAG-2-deficient) cell DNA and buffer (lane C). DNA samples from panel C were amplified with primers specific for a nonrearranging genomic locus, demonstrating the presence of DNA in all samples. (D) Ethidium bromide-stained agarose gel of these control amplifications. Lanes 1 to 4 correspond to samples 1 to 4 in panel C, lanes 5 to 8 correspond to samples 6 to 9 in panel C, and lane 9 is a buffer-only control amplification. Lanes 1 to 6 of panels A and B were shown to contain equivalent amounts of DNA by a similar method (data not shown). ce, coding ends; se, signal ends.

Article Snippet: Some plug DNA samples were subjected to T4 DNA polymerase treatment by incubating 40 μl of plug in an 80-μl reaction mixture with manufacturer’s buffer (Life Technologies), 5 U of T4 DNA polymerase, and 100 μM deoxynucleoside triphosphates at 37°C for 1 h. Other plugs were treated with various amounts of mung bean nuclease (BRL) under conditions previously reported by Zhu and Roth ( ).

Techniques: Polymerase Chain Reaction, Amplification, Staining, Agarose Gel Electrophoresis

Journal: Molecular and Cellular Biology

Article Title: Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

doi:

Figure Lengend Snippet: Length heterogeneity of amplified coding ends and joints. D H , J H 1, J H 2, V κ , and J κ 1 coding ends (ce) (A, B, C, E, and F) and DJ H and VJ κ joints (D and G) were amplified from T4 polymerase-treated thymus or induced 103 bcl2/4 cell DNA. The amplified fragments were gel purified and end labeled with [γ- 32 P]ATP by using T4 DNA kinase. Labeled fragments were electrophoresed on 6% denaturing polyacrylamide gels alongside DNA sequencing ladders used as size markers. The diagrams adjacent to each coding-fragment gel image indicate the sequence position based on these comigrating sequence markers. Position zero in the diagrams corresponds to the full-length coding end (i.e., the junction between the RSS and the coding segment), positive numbers indicate coding-end deletions, and negative numbers indicate longer-than-full-length coding-end lengths. The diagrams adjoining DJ H and VJ κ joints indicate successive nucleotide lengths. In panel E, the lane labeled Vκ mkr contains a radiolabeled amplification product of genomic DNA demonstrating the length heterogeneity of intact V κ genes (see the text).

Article Snippet: Some plug DNA samples were subjected to T4 DNA polymerase treatment by incubating 40 μl of plug in an 80-μl reaction mixture with manufacturer’s buffer (Life Technologies), 5 U of T4 DNA polymerase, and 100 μM deoxynucleoside triphosphates at 37°C for 1 h. Other plugs were treated with various amounts of mung bean nuclease (BRL) under conditions previously reported by Zhu and Roth ( ).

Techniques: Amplification, Purification, Labeling, DNA Sequencing, Sequencing

Journal: Applied and Environmental Microbiology

Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase

doi: 10.1128/AEM.68.12.6146-6151.2002

Figure Lengend Snippet: Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or T4 DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.

Article Snippet: After the unidirectionally reassembled DNA fragments were purified, they were blunt-ended on both ends with T4 DNA polymerase or S1 nuclease and then sticky-ended on the 3′ end with Sal I. Fragments in the range of about 500 to 960 bp were isolated by preparative agarose gel electrophoresis and cloned into pSTV28.

Techniques: Construct, Agarose Gel Electrophoresis, Ligation, Expressing, Plasmid Preparation, Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: The C-terminal Domain of the DNA Polymerase Catalytic Subunit Regulates the Primase and Polymerase Activities of the Human DNA Polymerase α-Primase Complex *

doi: 10.1074/jbc.M114.570333

Figure Lengend Snippet: Same efficiency of the extension of DNA and RNA primers on hetero-homopolymeric hybrid and heteropolymeric DNA templates by the p180ΔN-core. For control of the full extension of the primers, we used reactions with T4 DNA polymerase, which robustly

Article Snippet: T4 DNA polymerase (Promega Corporation; stock concentration 25 n m ) was used as a control for the primer extensions on hetero-DNA and RNA.

Techniques:

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins 1

doi: 10.1007/978-1-59745-196-3_7

Figure Lengend Snippet: LIC procedure using pMCSG vectors. All MCSG vectors contain an Ssp I site (AATATT) positioned immediately after the sequence encoding the TEV protease recognition site. Cleavage with Ssp I (a blunt cutter) followed by treatment with T4 DNA polymerase in

Article Snippet: dCTP (100 mM) (Promega cat. no. U1221) Dithiothreitol (DTT, 100 mM), molecular biology grade (Sigma cat. no. D-9779) T4 DNA polymerase, LIC-qualified (Novagen cat. no. 70099) 10× T4 polymerase buffer (included with polymerase)

Techniques: Sequencing

Journal: Journal of Virology

Article Title: Transgenically Expressed T-Rep of Tomato Yellow Leaf Curl Sardinia Virus Acts as a trans-Dominant-Negative Mutant, Inhibiting Viral Transcription and Replication

doi: 10.1128/JVI.75.22.10573-10581.2001

Figure Lengend Snippet: Slowly migrating DNAs are converted into ocDNA by Taq (A) or T4 (B) DNA polymerase treatment. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slowly migrating viral DNAs are indicated with an asterisk (∗). (A) TNAs were extracted from wt protoplasts at 72 h posttransfection with pTOM6 alone (the two lanes on the right) or together with pTOM100C4(−) or pTOM100NT and analyzed directly (−) or following incubation with Taq DNA polymerase for the time indicated below. (B) TNAs were extracted from wt or transgenic (102.22) protoplasts at 72 h posttransfection with pSP97 (TYLCSV-ES[1]) and analyzed following a 1-h incubation with (+) or without (−) T4 DNA polymerase. Lane C, TNAs from a TYLCSV-infected tomato plant digested with Bgl II to show migration of linear DNA.

Article Snippet: Reactions were carried out at 37°C for 30 min in a final volume of 20 μl containing 400 ng of TNAs, 100 μM each dNTP, and 2 U of T4 DNA polymerase (Boehringer Mannheim) in the incubation buffer supplied, then the concentration of each dNTP was brought to 200 μM, and incubation was prolonged for another 30 min.

Techniques: Incubation, Transgenic Assay, Infection, Migration

Journal: Nature

Article Title: Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders

doi: 10.1038/nature25449

Figure Lengend Snippet: Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

Article Snippet: After the NT2 wash, DDX21-bound RNA–protein complexes were dephosphorylated with T4 PNK (NEB, catalogue number M0210) for 30 min in an Eppendorf Thermomixer at 37 °C, 15 s at 1,400 r.p.m., 90 s rest in a 30 μl reaction, pH 6.5, containing 10 units of T4 PNK, 0.1 μl SUPERase-IN, and 6 μl of PEG-400 (16.7% final).

Techniques: Inhibition, Immunofluorescence, Staining