Journal: BMC Veterinary Research

Article Title: First isolation and characterization of Brucella microti from wild boar

doi: 10.1186/s12917-015-0456-z

Figure Lengend Snippet: Results of upgraded “Bruce- and Suis-ladder” PCRs of HUN-Bmi-01 strain. left side Bruce-ladder v2.0 PCR with 1682, 1071, 774, 587, 510, 450, 272, and 152 bp amplicons. M: 100 bp DNA marker, S: B. suis , C: B. canis , O: B. ovis , A: B. abortus . right side Suis-ladder PCR with 774 and 197 bp amplicons. M: 100 bp DNA marker, S1: B. suis biovar. 1, S2: B. suis biovar. 2

Article Snippet: The barcoded library DNA samples were column purified using the Gel/PCR DNA Fragments Extraction kit (Geneaid Biotech Ltd., Taipei, Taiwan).

Techniques: Polymerase Chain Reaction, Marker

Journal: Nucleic Acids Research

Article Title: Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event

doi: 10.1093/nar/gkx171

Figure Lengend Snippet: Fe 2+ or Mn 2+ can replace Mg 2+ as a cofactor for Deep Vent (exo-) DNA polymerase. Reaction mixtures with Fe 2+ or Mg 2+ or Mn 2+ or no divalent cation were analyzed for amount of reactants and products at every other PCR cycle for 24 cycles. Reactant consumption and product yields are identical within the uncertainty of this experiment among ( A ) Fe 2+ , ( B ) Mg 2+ or ( C ) Mn 2+ . ( D ) The divalent-minus controls, which lack divalent cations, did not generate product. Neither did the ‘minus template’ negative control reactions (far left lanes of each gel), taken through 24 cycles, generate product.

Article Snippet: The enzyme was heat inactivated at 80°C for 20 min and linearized DNA purified with an IBI Scientific Gel/PCR DNA Fragment Extraction kit.

Techniques: Polymerase Chain Reaction, Negative Control

Journal: EMBO Molecular Medicine

Article Title: IGSF10 mutations dysregulate gonadotropin‐releasing hormone neuronal migration resulting in delayed puberty

doi: 10.15252/emmm.201606250

Figure Lengend Snippet: Flowchart of exome sequencing filtering outcomes Whole exome sequencing was initially performed on DNA extracted from the peripheral blood leukocytes of 111 individuals from the 18 most extensive families from our cohort (76 with DP and 35 controls). The exome sequences were aligned to the UCSC hg19 reference genome. Picard tools and the genome analysis toolkit were used to mark PCR duplicates, realign around indels, recalibrate quality scores, and call variants. Variants were analyzed further and filtered for potential causal variants using filters for quality control, predicted functional annotation, minor allele frequency (MAF), segregation with trait, variants in multiple families, and biological relevance (see Materials and Methods and Appendix Table S1 for further information on filtering criteria). Targeted exome sequencing using a Fluidigm array of 28 candidate genes identified post‐filtering was then performed in a further 42 families from the same cohort (288 individuals, 178 with DP and 110 controls). Variants post‐targeted resequencing were filtered using the same criteria as the whole exome sequencing data. Rare variant burden testing was performed for all genes selected for targeted resequencing, in order to rank candidate genes post‐targeted resequencing. A multiple comparison adjustment was applied to the set of 28 P ‐values post hoc (Benjamini et al , 2001 ). Screening of 100 further cohort controls was via conventional Sanger sequencing. Functional annotation of the variants as described elsewhere in Materials and Methods. DP, delayed puberty. *data unpublished.

Article Snippet: PCR products were separated on an agarose gel, extracted from gel using HiYield Gel/PCR DNA Fragments Extraction Kit (RBC Bioscience, New Taipei City, Taiwan), and sequenced.

Techniques: Sequencing, Polymerase Chain Reaction, Functional Assay, Variant Assay

Journal: Nature Communications

Article Title: Nuclear moonlighting of cytosolic glyceraldehyde-3-phosphate dehydrogenase regulates Arabidopsis response to heat stress

doi: 10.1038/s41467-020-17311-4

Figure Lengend Snippet: Effect of GAPC on NF-YC10 binding to its target promoters. a PCR verification of the precipitated DNA. Chromatin immunoprecipitation (ChIP) was performed using anti-Flag antibody from heat-treated mesophyll protoplasts that were isolated from the GAPC -altered Arabidopsis lines indicated on the top and transfected with 35S:NF-YC10-Flag . Input DNA (ID; see Methods for details) and DNA precipitated with (+) or without (−) the antibody were used for PCR with primers specific to the promoters of genes indicated on the right. Mock, non-transfected; Ubq10 , ubiquitin10. b Quantification of the precipitated DNA. Quantitative PCR was performed with the DNA samples obtained from a . Values are average ± S.D. and shown as % of PCR product amplified from the input DNA. P values indicate significant difference from WT determined by two-tailed student’s t test ( n = 3 independent pools of protoplasts). Black dots represent individual data points. c NF-YC10 protein abundance. After transfection, total proteins were extracted from the protoplasts and immunoblotting was performed with anti-Flag antibody (top). Coomassie blue staining is shown for loading control (bottom).

Article Snippet: The sample was centrifuged at 5000 × g for 3 min and supernatant was incubated with 0.2 M NaCl at 65 °C for 6 h to reverse crosslinking, then incubated with 1 μg proteinase K at 37 °C for 1 h. DNA was purified using Gel/PCR DNA Fragments Extraction Kit (IBI Scientific) according to manufacturer’s instruction.

Techniques: Binding Assay, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Isolation, Transfection, Real-time Polymerase Chain Reaction, Amplification, Two Tailed Test, Staining

Journal: Nature Communications

Article Title: Nuclear moonlighting of cytosolic glyceraldehyde-3-phosphate dehydrogenase regulates Arabidopsis response to heat stress

doi: 10.1038/s41467-020-17311-4

Figure Lengend Snippet: Expression of heat-inducible genes in GAPC -altered Arabidopsis. Total RNA was extracted from 5-day-old seedlings of GAPC1 -OE ( a ), GAPC2 -OE ( b ), and gapc1gapc2 ( c ) treated at 37 °C for 5 h and quantitative RT-PCR was performed with gene-specific primers. Values are average ± S.D. and shown as fold change to WT (dashed line). P values indicate significant difference from WT determined by two-tailed student’s t test ( n = 3 independent groups of > 10 seedlings). Black dots represent individual data points. Hsf, heat shock transcription factor; Hsp, heat shock protein; At1g75860, DNA ligase; At4g36010, pathogenesis-related thaumatin superfamily protein; LFG4, LIFEGUARD4 (Bax inhibitor-1 family protein); EGY3, ETHYLENE-DEPENDENT GRAVITROPISM-DEFICIENT AND YELLOW-GREEN-LIKE3; CLPB3, CASEIN LYTIC PROTEINASE B3; FBS1, F-BOX STRESS INDUCED1; SAP10, STRESS-ASSOCIATED PROTEIN10; DREB2C, DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2C; At1g75960, AMP-dependent synthetase and ligase family protein; ACS7, 1-AMINO-CYCLOPROPANE-1-CARBOXYLATE SYNTHASE7.

Article Snippet: The sample was centrifuged at 5000 × g for 3 min and supernatant was incubated with 0.2 M NaCl at 65 °C for 6 h to reverse crosslinking, then incubated with 1 μg proteinase K at 37 °C for 1 h. DNA was purified using Gel/PCR DNA Fragments Extraction Kit (IBI Scientific) according to manufacturer’s instruction.

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Binding Assay