Journal: Journal of Insect Science

Article Title: Rapid Identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) Using Ribosomal RNA Internal Transcribed Spacer 1

doi: 10.1093/jisesa/iev137

Figure Lengend Snippet: Derivative dissociation plots produced by different reagents using one microliter of lysate produced by homogenizing one H. armigera leg with 24 H. zea legs in 625 μl of modified squish buffer. (A) Amplification reactions made with KAPA SYBR Fast Master Mix (purple) and NEB LongAmp Taq polymerase and buffer with 2.5 mM MgCl2 (Red). Peaks representing 18S rRNA control amplicon and species-specific amplicons of H. armigera and H. zea are present in all reactions, but melting temperatures were different in two reagents. (B) An enlarged view of the derivative dissociation plot produced by amplification reactions with NEB LongAmp Taq polymerase and buffer. Although variations in dissociation plots between samples were observed, the 3-peak dissociation plot was clearly identifiable.

Article Snippet: LongAmp Taq polymerase and reaction buffer with 2.5 mM MgCl2 (New England Biolabs) produced dissociation curves with lower intensity than ready-to-use master mixes but sufficient to detect H. armigera in 1:24 ratio of H. armigera : H. zea tissue lysate ( a and b).

Techniques: Produced, Modification, Amplification

Journal: Biological Procedures Online

Article Title: Rapid and simple comparison of messenger RNA levels using real-time PCR

doi: 10.1251/bpo114

Figure Lengend Snippet: MgCl2: Melt® results.

Article Snippet: Each sample consisted of: 50 ng cDNA, 3 mM MgCl2 , 200 μM dNTP, 500 nM of primers, 2 μl of 10X PCR buffer, 0.1 unit of Taq polymerase and SYBR® Green (Molecular Probe, Eugene, OR; 1/30 000 dilution), in a reaction volume of 20 μl.

Techniques:

Journal: Biological Procedures Online

Article Title: Rapid and simple comparison of messenger RNA levels using real-time PCR

doi: 10.1251/bpo114

Figure Lengend Snippet: Optimization of MgCl2 concentration in samples.

Article Snippet: Each sample consisted of: 50 ng cDNA, 3 mM MgCl2 , 200 μM dNTP, 500 nM of primers, 2 μl of 10X PCR buffer, 0.1 unit of Taq polymerase and SYBR® Green (Molecular Probe, Eugene, OR; 1/30 000 dilution), in a reaction volume of 20 μl.

Techniques: Concentration Assay

Journal: The EMBO Journal

Article Title: DNA polymerase mu (Pol ?), homologous to TdT, could act as a DNA mutator in eukaryotic cells

doi: 10.1093/emboj/19.7.1731

Figure Lengend Snippet: Fig. 6. Pol μ-catalysed misinsertion at the four template bases. The four template–primer structures used, which differ only in the first template base (outlined), are indicated on the left. The single-stranded oligonucleotide corresponding to the primer strand was assayed in parallel as a control of DNA-independent nucleotide insertion. Mg 2+ -activated nucleotide insertion on each 5′–labelled DNA substrate (3.2 nM) was analysed in the presence of either the complementary nucleotide (10 μM) or each of the three incorrect dNTPs (100 μM), as described in Materials and methods. Mn 2+ -activated nucleotide insertion was assayed with each of the four dNTPs (0.1 μM). After incubation for 15 min at 30°C in the presence of 20 ng of human Pol μ, extension of the 5′–labelled (*) strand was analysed by electrophoresis in an 8 M urea–20% polyacrylamide gel and autoradiography.

Article Snippet: The PCRs were performed in a total volume of 10 μl, using 25 ng of template, 5 μM primers h2MAPs and h2MAPas, 1.25 mM MgCl2 , 0.2 mM dNTPs and 0.025 U of Taq DNA polymerase (Gibco-BRL) in the buffer supplied by the manufacturer.

Techniques: Incubation, Electrophoresis, Autoradiography

Journal: The EMBO Journal

Article Title: DNA polymerase mu (Pol ?), homologous to TdT, could act as a DNA mutator in eukaryotic cells

doi: 10.1093/emboj/19.7.1731

Figure Lengend Snippet: Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and dTTP, in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.

Article Snippet: The PCRs were performed in a total volume of 10 μl, using 25 ng of template, 5 μM primers h2MAPs and h2MAPas, 1.25 mM MgCl2 , 0.2 mM dNTPs and 0.025 U of Taq DNA polymerase (Gibco-BRL) in the buffer supplied by the manufacturer.

Techniques: Inhibition, Incubation, Concentration Assay

Journal: AoB Plants

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline

doi: 10.1093/aobpla/pls033

Figure Lengend Snippet: Amplification of local Alexandrium strains ACC01, ACC02 and ACC07 using species - specific primers; A. tamarense (lanes 1, 2 and 3) and A. catenella (lanes 4, 5 and 6). M = 100-bp DNA size marker. Species-specific amplification in the rDNA region using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller™ 100-bp DNA ladder was used for size estimation of amplified fragments.

Article Snippet: All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume.

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

Journal: AoB Plants

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline

doi: 10.1093/aobpla/pls033

Figure Lengend Snippet: Alexandrium tamarense microsatellite amplifications of the three local strains with (A) specific primers ATB8 (lanes 1, 2 and 3) and ATD8 (lanes 4, 5 and 6). Lanes 7 and 8 correspond to controls with primers ATB8 without DNA. (B) Specific amplification with primers ATB1 (lanes 1, 2 and 3) and ATF11 (lanes 6, 7 and 8). Lanes 4–5 and 9–10 are controls without DNA for primer sets ATB1 and ATF11, respectively. M = 100-bp DNA size marker. Species-specific microsatellite amplifications using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller TM 100-bp DNA ladder was used for size estimation of amplified fragments.

Article Snippet: All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume.

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

Journal: eLife

Article Title: PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins

doi: 10.7554/eLife.50416

Figure Lengend Snippet: PPM1H dephosphorylates Rab10 in vitro. ( A ) The indicated amounts of recombinant wild-type and mutant PPM1H (with a His-Sumo N-terminal tag, expressed in E. coli ) were incubated in vitro with 2.5 µg pT73 ~60% phosphorylated Rab10[1–181, GDP bound] for 30 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.0 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by phos-tag gel electrophoresis that separates phosphorylated and dephosphorylated Rab10. Gel was stained with Instant Blue Coomassie. Bands corresponding to phosphorylated and non-phosphorylated Rab10 are marked with open (○) and closed (●) circles respectively. ( B ) As in ( A ) except that a time-course assay was performed using 2.5 µg pT73 phosphorylated Rab10[1–181, GDP bound] and 40 ng wild-type or mutant PPM1H for the indicated times. ( C ) As in ( A ) except that PPM1J was assessed. ( D ) As in ( A ) except and PPM1M was assessed.

Article Snippet: The protein was eluted with 4 × 1 resin volume of elution buffer (30 mM Tris pH 7.5, 3% (by vol) glycerol, 0.4 M imidazole, 8 mM 2-mercaptoethanol, 1.2 mM MgCl2 , 0.02% (by vol) Brij35, 0.6 µM GTP-gamma-S) and dialyzed against 50 mM Tris pH 7.5, 10% (by vol) glycerol, 250 mM NaCl, 14 mM 2-mercaptoethanol, 2 mM MgCl2 , 0.6 µM GTP-gamma-S in the presence of 60 Units of Thrombin (Sigma-Aldrich T1063-1KU).

Techniques: In Vitro, Recombinant, Mutagenesis, Incubation, Nucleic Acid Electrophoresis, Staining

Journal: Frontiers in Microbiology

Article Title: Geomicrobiological Features of Ferruginous Sediments from Lake Towuti, Indonesia

doi: 10.3389/fmicb.2016.01007

Figure Lengend Snippet: Multiple profiles established on short sediment cores. For consistency, results for the three different sites are displayed vertically according to their water depths. (A) From left to right: Total organic carbon and total carbon (wt%); molar C org /N ratio measured in bulk sediments; calcium, magnesium, chloride, and sulfate concentrations (μM) measured in the pore water; potential sulfate reduction rates (nmol × cm -3 day -1 ) obtained after 24 h incubation experiments; ammonium concentrations (μM) in the pore water measured by cell dialysis. (B) From left to right: Cell counts in log scale (log 10 cells × cm -3 ); concentrations of extracellular DNA (gray dots) and total DNA (black squares) (μg × g wet sediment -1 ), with distance between the two curves corresponding to intracellular DNA concentrations; Shannon index established from bacterial and archaeal DGGE gel features, with eDNA (gray dots) and iDNA (black squares) displayed separately; range-weighted richness index for bacterial DNA (square) and archaeal DNA (dots), with eDNA (gray) and iDNA (black) displayed separately.

Article Snippet: PCR was performed with 2.5 μL of DNA template, 12.5 μL of MangoMix (Bioline, Life Science Company, London, UK), 0.5 μmol L-1 of each of the primers, 1 μL of MgCl2 (25 mM) and 8 μL of Ultra-pure 18.2 Mω PCR Water (Bioline).

Techniques: Incubation, Denaturing Gradient Gel Electrophoresis

Journal: Biochemistry

Article Title: Isozyme Specific Allosteric Regulation of Human Sulfotransferase 1A1

doi: 10.1021/acs.biochem.6b00401

Figure Lengend Snippet: EGCG binding to the E·(PAP)2 Complex. A.) The Binding-Reaction Progress Curve . Binding-induced fluorescence changes were monitored using a stopped-flow fluorimeter (λ ex = 290 nm, λ em ≥ 325 nm (cutoff filter). Reactions were initiated by rapidly mixing (1:1 v/v) a solution containing SULT1A1 (0.25μM, active sites), PAP (0.50 mM), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.5, 25 ± 2°C with a solution that was identical except that it lacked SULT1A1 and contained EGCG. The curve through the data (red line) is the behavior predicted by the best fit to a double-exponential model. The insert shows the residuals from a single- (black) and doubleexponential (red) fit. The single-exponential model deviates substantially from the data, while the bi-phasic model provides an excellent fit. B.) k obs vs [EGCG] for the First (Fast) Phase . kobs values were obtained at the four indicated EGCG concentrations under the conditions given in Panel A. Each value is the average of three independent determinations. The reactions were pseudo-first-order in EGCG in all cases. C.) EGCG Dissociation from E·(PAP) 2 ·(EGCG) 2 Complex . A pulse solution containing SULT1A1 (20 μM, monomer), EGCG (24 μM (4.0 μM free, 100 × Kd)), and PAP (500 μM, 16 ▪ K d low affinity site ), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.5, 25 ± 2°C, was diluted 200:1 into a chase solution containing 75 μM 4-hydroxytamoxifen (TAM, 100 ▪ K d cap open form ), PAP (0.50 mM, 16 ▪ K d low affinity site ), NaPO4 (50 mM), MgCl2 (5.0 mM), pH 7.5, 25 ± 2°C. The final EGCG concentration is 0.12 μM (0.20 ▪ K d cap open form ). Dissociation was monitored by a change in SULT1A1 fluorescence (λ ex = 290 nm, λ em = 370 nm). Three progress curves (black dots) are superposed. The curve through the data is the behavior predicted by a best-fit, single-exponential model.

Article Snippet: Ampicillin, HEPES, KCl, KOH, MgCl2 , NaCl and phenylmethylsulfonyl fluoride (PMSF) were purchased from Fisher Scientific.

Techniques: Binding Assay, Fluorescence, Flow Cytometry, Concentration Assay

Journal: Nature Communications

Article Title: RIP2 filament formation is required for NOD2 dependent NF-κB signalling

doi: 10.1038/s41467-018-06451-3

Figure Lengend Snippet: Full-length RIP2 forms filaments in vitro. a Domain organization of the MBP-RIP2fl construct used for expression and purification of recombinant RIP2fl from sf21 insect cells. b 12.5 % SDS-PAGE showing recombinant RIP2fl at consecutive purifications steps. c Typical size exclusion chromatography profile for tagged (blue) and tag-free RIP2fl (black). d In vitro kinase activity of RIP2fl. Complete auto-phosphorylation is achieved after 15 min. e – g Negative-stain micrographs of ( e ) VV RIP2fl, ( f ) RIP2fl and ( g ) RIP2fl with added ATP and magnesium chloride. Scale bars are 100 nm. EAR: eluate from amylose resin, C: sample after tag cleavage as loaded on size-exclusion chromatography, VV: size exclusion chromatography void volume, Min: minutes

Article Snippet: Cells from 1.2 L culture resuspended in 20 mM Tris pH 8, 20 mM NaCl, 2 M urea, 2 mM β-Me, 1 mM Pefabloc (Sigma), 5 mM MgCl2 , and 15 µL Benzonase (Merck) were disintegrated by high pressure (Microfluidizer LM10, Microfluidics).

Techniques: In Vitro, Construct, Expressing, Purification, Recombinant, SDS Page, Size-exclusion Chromatography, Activity Assay, Staining

Journal: Nature Communications

Article Title: RIP2 filament formation is required for NOD2 dependent NF-κB signalling

doi: 10.1038/s41467-018-06451-3

Figure Lengend Snippet: Full-length RIP2 filaments are promoted by nucleotide binding. a – c  Negative-stain micrographs of MBP-RIP2fl filaments obtained from ( a ) MBP-RIP2fl alone, ( b ) MBP-RIP2fl plus ATP dissolved in magnesium chloride buffer, ( c ) MBP-RIP2fl plus magnesium, 5 mM nucleotide dissolved in buffer containing magnesium chloride: ( d ) AMP, ( e ) ADP and ( f ) AMPPCP. Scale bars are 100 nm

Article Snippet: Cells from 1.2 L culture resuspended in 20 mM Tris pH 8, 20 mM NaCl, 2 M urea, 2 mM β-Me, 1 mM Pefabloc (Sigma), 5 mM MgCl2 , and 15 µL Benzonase (Merck) were disintegrated by high pressure (Microfluidizer LM10, Microfluidics).

Techniques: Binding Assay, Staining

Journal: Scientific Reports

Article Title: Concentration Dependent Ion-Protein Interaction Patterns Underlying Protein Oligomerization Behaviours

doi: 10.1038/srep24131

Figure Lengend Snippet: SNAP25 in native plasma membrane sheets shows biphasic clustering in response to divalent ions. PC12 plasma membrane sheets incubated at 37 °C for 10 min with the indicated concentrations of calcium, strontium, barium or magnesium chloride, fixed, immunostained for SNAP25 and analysed with epifluorescence or superresolution STED microscopy. ( a ) Membrane sheets incubated with 0, 10, or 1000 mM CaCl 2 . Epifluorescence recordings from the dye TMA-DPH (left; documenting the integrity of the membranes) and the immunostaining (middle; overviews are shown at the same, magnified insets at arbitrary scaling). Right, STED micrographs of SNAP25 immunofluorescence to which the “red hot” look up table was applied which displays increasingly brighter pixel intensities applying a colour code from black to red to yellow to white. ( b ) SNAP25 clustering was quantified by calculating the relative standard deviation (rel. SD) of the immunostaining pattern, normalized to the baseline condition which contained no divalent cations. Values are means ± s.e.m. ( c ) SNAP25 cluster density (means ± s.e.m.) resolved by superresolution STED microscopy. Cluster size was similar under all conditions ( Supplementary Fig. 4 ).

Article Snippet: Microscale thermophoresis (MST) Titration series with 10 μM SNAP25 and CaCl2 or MgCl2 (Carl Roth) concentrations varying between 0 and 1000 mM were prepared in TBS containing 0.1% [v/v] Pluronic F-127 (Sigma, St. Louis, MO).

Techniques: Incubation, Microscopy, Immunostaining, Immunofluorescence, Standard Deviation

Journal: The Journal of Experimental Medicine

Article Title: Chromosomal reinsertion of broken RSS ends during T cell development

doi: 10.1084/jem.20070583

Figure Lengend Snippet: Gel analysis of LM-TECA PCR products. 10 0.2-μl aliquots (lanes 1–10) of captured wild-type thymus DNA were subjected to PCR analysis for J-sided RSS transposition events. The filled arrow indicates the 629-bp amplification product from the unrearranged TCRβ locus that is caused by the restriction site used to fragment the genomic DNA ( Fig. 1 A , vertical gray arrow). Individual bands were excised from the gel and subjected to DNA sequence analysis. The open arrows indicate two RSS insertion events, whereas the other discrete bands correspond to random breaks along the germline TCRβ locus. Neg, a negative PCR control consisting of 63–12 cell DNA; mkr, MspI-digested pBR322.

Article Snippet: Plugs were rinsed with TE (10 mM Tris-HCl, pH 8.0, and 0.1mM EDTA) and digested genomic DNA was mixed with 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , and 0.001% gelatin), 0.2 mM dNTPs, 175 pmol of a 5′-biotinylated oligonucleotide (Table S1, available at http://www.jem.org/cgi/content/full/jem.20070583/DC1 ), and 20 U of a hot-start Taq polymerase (JumpStart; Sigma-Aldrich) in a final volume of 800 μl.

Techniques: Polymerase Chain Reaction, Amplification, Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Chromosomal reinsertion of broken RSS ends during T cell development

doi: 10.1084/jem.20070583

Figure Lengend Snippet: The frequency of d - or J-side RSS insertion into the b196 hotspot in wild-type and mutant mice. 85 1-μg (∼166,000 genome) samples of thymus or spleen DNA from the indicated mice were analyzed by PCR for either D or J RSS hotspot insertion events. Frequency was calculated according to the Poisson distribution. Error bars on the cR2 Thy and WT Thy samples indicate analysis of independent sets of 85 PCR assays from each of 3 different animals.

Article Snippet: Plugs were rinsed with TE (10 mM Tris-HCl, pH 8.0, and 0.1mM EDTA) and digested genomic DNA was mixed with 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , and 0.001% gelatin), 0.2 mM dNTPs, 175 pmol of a 5′-biotinylated oligonucleotide (Table S1, available at http://www.jem.org/cgi/content/full/jem.20070583/DC1 ), and 20 U of a hot-start Taq polymerase (JumpStart; Sigma-Aldrich) in a final volume of 800 μl.

Techniques: Mutagenesis, Mouse Assay, Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: Chromosomal reinsertion of broken RSS ends during T cell development

doi: 10.1084/jem.20070583

Figure Lengend Snippet: Identifying the second end of a Jβ1.1 RSS fusion to chromosome 12 (event EL3-1). (A) Diagram of the Jβ1.1 RSS-12 (open triangle) insertion into chromosome 12, which includes a 7-nt N region (sequence shown). The hypothetical structure of the other end of this transposed fragment is indicated (Dβ1 RSS-23; filled triangle) along with the nested set of PCR primers used. A negative image of the gel analysis of this PCR assay is shown below the diagram, containing the predicted 123-bp product. Lanes labeled Thymus-1 DNA are PCR reactions programmed with independent DNA samples from the same thymus. Lane 63–12 is DNA from a RAG2 −/− pro-B cell line. (B) PhosphorImages of Southern blotted PCR products from a thymus sample and a set of spleen samples from the same mouse (indicated as “1”), along with four thymus samples from different mice (top) ( 2 – 5 ), and a set of twofold serial dilutions of thymus DNA and a spleen sample (Spl-1) from animal 1 analyzed for the chr12-Dβ1-RSS23 insertion (bottom). (C) Diagram of the presumptive insertion event and a PCR strategy to determine if the characterized Dβ1 RSS end is contiguous with the Jβ1 RSS end. The negative image of an ethidium-stained agarose gel analysis of PCR products (740 bp) from DNA purified from four aliquots of animal 1's thymus and two aliquots of animal 1's spleen is shown. The identity of this PCR product was confirmed by DNA sequencing.

Article Snippet: Plugs were rinsed with TE (10 mM Tris-HCl, pH 8.0, and 0.1mM EDTA) and digested genomic DNA was mixed with 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , and 0.001% gelatin), 0.2 mM dNTPs, 175 pmol of a 5′-biotinylated oligonucleotide (Table S1, available at http://www.jem.org/cgi/content/full/jem.20070583/DC1 ), and 20 U of a hot-start Taq polymerase (JumpStart; Sigma-Aldrich) in a final volume of 800 μl.

Techniques: Sequencing, Polymerase Chain Reaction, Labeling, Mouse Assay, Staining, Agarose Gel Electrophoresis, Purification, DNA Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Chromosomal reinsertion of broken RSS ends during T cell development

doi: 10.1084/jem.20070583

Figure Lengend Snippet: A hotspot for Dβ1 RSS transposition (event b196). (A, top) Diagram of the captured Jβ1.1-RSS12 (open triangle) integration into chromosome 1 showing the PCR strategy used to assay for the corresponding Dβ1-RSS23 (filled triangle) integration. (middle) The negative image of an ethidium-stained agarose gel analysis of PCR products (189 bp) from 6 thymus DNA samples from the same animal. (bottom) Results of DNA sequence analysis with the junction sequence shown. Lane 63–12 is a RAG-2 –deficient pro-B cell line. (B) PCR analysis of the Dβ1-RSS23 insertion into chromosome 1 using two aliquots of thymus DNA from each of four different mice ( 1 – 4 ). The DNA sequence at the chromosome 1–Dβ1 RSS junction is indicated below each amplified product. Nucleotides in parentheses are N regions. (C) PCR analysis of Dβ1-RSS23 insertion into chromosome 1 in two separate aliquots of thymus (Thy), spleen (Spl), and bone marrow (BM) DNA from three additional mice ( 5 – 7 ). β-Globin control PCR products (labeled β-G) show that similar amounts of DNA were amplified in each case. The chromosome 1—Dβ1-RSS23 products were sequenced as indicated. Overlapping sequences denote multiple sequences present in a single PCR band.

Article Snippet: Plugs were rinsed with TE (10 mM Tris-HCl, pH 8.0, and 0.1mM EDTA) and digested genomic DNA was mixed with 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , and 0.001% gelatin), 0.2 mM dNTPs, 175 pmol of a 5′-biotinylated oligonucleotide (Table S1, available at http://www.jem.org/cgi/content/full/jem.20070583/DC1 ), and 20 U of a hot-start Taq polymerase (JumpStart; Sigma-Aldrich) in a final volume of 800 μl.

Techniques: Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Sequencing, Mouse Assay, Amplification, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Chromosomal reinsertion of broken RSS ends during T cell development

doi: 10.1084/jem.20070583

Figure Lengend Snippet: The LM-TECA assay for TCRb locus RSS-fragment insertion. (A) Diagram of the germline TCRβ locus (top) and its various recombination intermediates (middle) and alternative products (bottom two lines). Triangles indicate recombination signal sequences (open triangle, RSS-12; filled triangle, RSS-23). The approximate locations of restriction sites used in LM-TECA are indicated by vertical gray arrows. (B) RSS-ended fragment excised from the TCRβ locus (thin line) shown with its reactive 39′OH groups inserting into a target DNA (thick line) elsewhere in the genome, resulting in an RSS fragment insertion event. (C) The LM-TECA method. B→ indicates a 5′ biotin-labeled primer, the white square indicates an oligonucleotide linker, and arrows indicate PCR primers (open arrow, linker primer; filled arrow, locus primer). (D) Strategy for amplification of the “other” end of an RSS fragment insertion or a balanced translocation. PCR primers are shown as arrows.

Article Snippet: Plugs were rinsed with TE (10 mM Tris-HCl, pH 8.0, and 0.1mM EDTA) and digested genomic DNA was mixed with 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , and 0.001% gelatin), 0.2 mM dNTPs, 175 pmol of a 5′-biotinylated oligonucleotide (Table S1, available at http://www.jem.org/cgi/content/full/jem.20070583/DC1 ), and 20 U of a hot-start Taq polymerase (JumpStart; Sigma-Aldrich) in a final volume of 800 μl.

Techniques: Labeling, Polymerase Chain Reaction, Amplification, Translocation Assay

Journal: The Journal of Experimental Medicine

Article Title: Chromosomal reinsertion of broken RSS ends during T cell development

doi: 10.1084/jem.20070583

Figure Lengend Snippet: Signal joints can undergo recombination with chromosomal RSSs. (A) Diagram of a strategy to detect insertion of a transfected recombination signal circle at a Vκ RSS. The arrows indicate PCR primers used to detect RSS insertion events. Triangles represent RSSs (hatched, TCRβ D or J; unfilled, Vκ RSS-12). (B) Agarose gel analysis of PCR assays (primers a and b) for RSS insertion in genomic DNA purified from cells transfected with wild-type (lane 1), RSS-23 mutant (lane 2), or N region–containing (lane 3) TCRβ signal circle plasmids. Arrow denotes the predicted ∼119-bp PCR products that were confirmed by DNA sequence analysis. (C) Agarose gel analysis of PCR assays (primers c and d) using DNA purified from cells transfected with wild-type (lanes 1 and 2) and N region–containing (lanes 3 and 4) signal joint plasmids. PCR products were either digested (+) or not (-) with restriction endonuclease ApaL1, which cleaves precise signal joints. Filled arrow, predicted 142-bp PCR product; open arrow, 115-bp ApaLI-cleaved product.

Article Snippet: Plugs were rinsed with TE (10 mM Tris-HCl, pH 8.0, and 0.1mM EDTA) and digested genomic DNA was mixed with 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , and 0.001% gelatin), 0.2 mM dNTPs, 175 pmol of a 5′-biotinylated oligonucleotide (Table S1, available at http://www.jem.org/cgi/content/full/jem.20070583/DC1 ), and 20 U of a hot-start Taq polymerase (JumpStart; Sigma-Aldrich) in a final volume of 800 μl.

Techniques: Transfection, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Purification, Mutagenesis, Sequencing