Journal: Genome Research

Article Title: Guide Positioning Sequencing identifies aberrant DNA methylation patterns that alter cell identity and tumor-immune surveillance networks

doi: 10.1101/gr.240606.118

Figure Lengend Snippet: Guide Positioning Sequencing (GPS) detects genome-wide DNA methylation accurately with high coverage rate. ( A ) Schematic of GPS workflow for DNA methylation detection. The gray line represents original DNA sequence, and the orange line represents DNA treated by T4 DNA polymerase, which replaces cytosine with 5′-methylcytosine at 3′ end of DNA fragment. The solid circle (●) represents methylated cytosine, and the open circle (○) represents unmethylated cytosine, whereas the triangle (Δ) represents thymine. Blue and green short lines represent the NGS linker. Read1 represents the bisulfite-converted 5′ end of fragments, whereas Read2 represents the 3′ end of fragments, which is the same as the genome sequence due to 5′-methylcytosine replacement. ( B ) The accurate alignment rate of Bowtie 2 and GPS is obviously higher than that in BSMAP based on simulated data: (***) P

Article Snippet: Thirty units of T4 DNA polymerase (New England BioLabs, M0203L) was used to perform 3′→5′ digestion of the DNA fragments for 100 min at 12°C followed by adding 10 µL dNTP mix which contained dATP, dTTP, dGTP, and 5′-methyl-dCTP nucleotide (final concentration 0.5 mM) and incubating for 30 min at 37°C.

Techniques: Sequencing, Genome Wide, DNA Methylation Assay, Methylation, Next-Generation Sequencing

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 Mammalian Cell Based FACS-Panning Platform for the Selection of HIV-1 Envelopes for Vaccine Development

doi: 10.1371/journal.pone.0109196

Figure Lengend Snippet: Schematic overview of the QL cloning procedure. An envelope gene or an envelope library is amplified with primers to introduce flanking Esp3I restriction sites enabling the generation of a 5′ NcoI and a 3′ Xho sitey (A; top). The envelope gene or an envelope library is incubated together with pQL9/11 in a one-tube reaction with Esp3I and T4-Ligase. Compatible “sticky-ends” (equally colored) can be ligated successfully, direct proper orientation and mediating resistance for further cleavage (A). Following transformation of CcdB sensitive bacteria, only recipients bearing a plasmid without CcdB are able to form colonies in the presence of ampicillin. (B) The lentiviral vector construct pQL9 comprises (i) 5′LTR (Long terminal repeat), (ii) EF1α (human promotor), (iii) GFP (marker gene), (iv) an IRES (internal ribosome entry site), (v) a CcdB positive selection marker [58] , and (vi) a 3′LTR sequence.

Article Snippet: Meanwhile a second reaction for the ligation was prepared. (II) 3 µL 10 mM ATP, 1 µL 10 x Tango Buffer, 1 µL 10 mM DTT, 1 µL T4-Ligase (NEB) addition of H2 0 to reach 10 µL.

Techniques: Clone Assay, Amplification, Introduce, Incubation, Transformation Assay, Plasmid Preparation, Construct, Marker, Selection, Sequencing

Journal: Nucleic Acids Research

Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes

doi: 10.1093/nar/gkx1249

Figure Lengend Snippet: ExoCET mechanism. ( A ) Juxtaposition of the 80-bp homology arms between the p15A-cm (chloramphenicol) vector and the 14-kb lux genomic segment is illustrated: (a) both homology arms were located at the termini; (b and c) one homology arm was located at a terminus and the other 1 kb from the other end; (d) both homology arms were 1 kb from each end. ( B ) Number of colonies obtained from ETgA, T4pol or ExoCET using the homology arm combinations (a–d) as indicated. Reaction conditions were the same as for Figure 1F . ( C ) Protein combinations as indicated expressed from pSC101 plasmids in GB2005 were tested for direct cloning of the 14-kb lux gene cluster using terminal homology arms and ExoCET conditions except for the omission of RecA (ETg); RecA and RecT (Eg), RecA and RecE (Tg) and all (pSC101-tet). Error bars, s.d.; n = 3. Corresponding DNA analyses are shown in Supplementary Figure S5 .

Article Snippet: In vitro assembly Ten micrograms of genomic DNA and 200 ng of 2.2-kb p15A-cm linear vector (1 μg of 8-kb linear BAC vector) were assembled in 20 μl reactions consisting of 2 μl of 10 × NEBuffer 2.1 and 0.13 μl of 3 U μl−1 T4pol (NEB, cat. no. M0203).

Techniques: Plasmid Preparation, Clone Assay

Journal: Nucleic Acids Research

Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes

doi: 10.1093/nar/gkx1249

Figure Lengend Snippet: Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).

Article Snippet: In vitro assembly Ten micrograms of genomic DNA and 200 ng of 2.2-kb p15A-cm linear vector (1 μg of 8-kb linear BAC vector) were assembled in 20 μl reactions consisting of 2 μl of 10 × NEBuffer 2.1 and 0.13 μl of 3 U μl−1 T4pol (NEB, cat. no. M0203).

Techniques: In Vitro, Clone Assay, Plasmid Preparation, Electroporation, Titration, Incubation, Standard Deviation

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: Nucleic Acids Research

Article Title: A novel and simple method for construction of recombinant adenoviruses

doi: 10.1093/nar/gkl449

Figure Lengend Snippet: The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.

Article Snippet: Cloning the foreign genes gfp and man into the donor plasmid using restriction enzyme Bsu36I and T4 DNA polymerase The gfp gene was amplified from pEGFP-1 (Clontech) by PCR.

Techniques: Polymerase Chain Reaction, Amplification, Ligation, Transformation Assay, Recombinant, Plasmid Preparation, Activity Assay

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: The Journal of Biological Chemistry

Article Title: UV Damage in DNA Promotes Nucleosome Unwrapping *

doi: 10.1074/jbc.M110.140087

Figure Lengend Snippet: UV damage does not affect NCP reconstitution with the 601 sequence. A , NCP reconstitution with UV-undamaged and -damaged DNA. The 147-bp 601 DNA containing both UV lesions and labeling were mixed with histone octamer at 2 m NaCl. The reconstitution was performed by stepwise salt dialysis, and the final NaCl concentration was 50 m m . The reconstituted products were resolved in 5% native polyacrylamide gel and stained with SYBR Gold. The 100-bp DNA markers are indicated on the left. B , presence of CPDs and 6-4PPs in UV-damaged DNA. The different UV-damaged DNA were blotted on the nitrocellulose and detected by lesion-specific antibodies. The same membranes were reprobed with 32 P-labeled DNA to show equal loading. C , Southern blot of the photoproduct yield of the UV-irradiated DNA fragment. The DNA was treated with or without photolyase prior to the T4 DNA polymerase ( pol ) digestion. The digested samples were blotted on the nylon membrane and probed with with 32 P-labeled DNA. D , quantification data of the photoproduct yield by Southern blots. The CPD signals were calculated by subtracting the total signals with the 6-4PPs signals. Three independent experiments were performed to show error bars .

Article Snippet: Briefly, samples were incubated with 2.5 units of T4 DNA polymerase-exonuclease (Fermentas) at 37 °C for 2 h. The reaction was stopped by heating at 65 °C for 10 min.

Techniques: Sequencing, Labeling, Concentration Assay, Staining, Southern Blot, Irradiation

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: BMC Genomics

Article Title: Multigene family isoform profiling from blood cell lineages

doi: 10.1186/1471-2164-3-22

Figure Lengend Snippet: Validation of Kir isoform typing with reporter enzyme ScrF I. Degenerate PCR with sub-family centred primers was performed on Kir subclone templates and products were T4 DNA polymerase end-labelled according to Materials and Methods . Diagnostic restriction digests were performed using ScrF I and an isoform specific duplex banding pattern generated consistent with the predicted banding patterns described in Figure 1 . Faint low MW fragments (

Article Snippet: T4 DNA Polymerase end-labelling of degenerate PCR products The products from the degenerate, sub-family centred PCR were excised and purified from agarose gels using a kit (Hybaid Ltd, Middlesex, UK) according to manufacturers protocol, and eluted into 20 μl elution solution.

Techniques: Polymerase Chain Reaction, Diagnostic Assay, Generated

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: 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 enzymology

Article Title: RNA-ID, a Powerful Tool for Identifying and Characterizing Regulatory Sequences

doi: 10.1016/bs.mie.2016.02.003

Figure Lengend Snippet: Preparation of vector and inserts for LIC. (A) A schematic of the vector pEKD1024 showing the major steps to generate single stranded DNA ends for LIC using T4 DNA polymerase and dGTP. Restriction enzyme sites are shaded and the nucleotide where the T4 DNA polymerase stops is indicated by white text over a dark background. (B, C, D) Schematics of various types of inserts that can be cloned into the vector pEKD1024. (B) A PCR amplified insert and the product after treatment with T4 DNA polymerase in the presence of dCTP. (C, D) Inserts formed with oligonucleotides. (C) Complete overlap of inserted sequences. (D) Partially randomized sequence with 13 nucleotide overlap.

Article Snippet: Incubate at 22 °C (room temperature) for 40 minutes, then inactivate the T4 DNA polymerase by incubating the reaction at 75 °C for 40 minutes.

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

Journal: Cancers

Article Title: Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

doi: 10.3390/cancers6031464

Figure Lengend Snippet: MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [ 14 ]. This origin was used for replication assays ( Figure 3 and Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun et al. [ 31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using T4 polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.

Article Snippet: The purified probe (100 ng) was then 5' labeled with [32 P-γ] ATP with T4 Polynucleotide Kinase (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s instructions.

Techniques: Sequencing, Clone Assay, Plasmid Preparation, Generated, Polymerase Chain Reaction, Amplification, Labeling, Western Blot, Purification, Mutagenesis, Affinity Purification, Negative Control

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