pbluescript (pbs) ii sk  (Agilent technologies)

Journal: PLOS ONE

Article Title: DAPE cloning with modified primers for producing designated lengths of 3’ single-stranded ends in PCR products

doi: 10.1371/journal.pone.0318015

Figure Lengend Snippet: (A) A pBluescript II SK+ vector was chosen as a PCR template for amplifying the indicated lengths of fragments. Both PCR products, depicted in blue and red, were combined and assembled into the intact pBluescript II SK+ vector using SLIC techniques. The black lines represent the 15 bp complementary sequences of each product. (B) A schematic diagram of phosphodiester and phosphorothioate (PT) bonds used in the preparation of primer synthesis. (C) Primers for amplifying the two different regions of pBluescript II SK+ in (A) were modified with a 5’ end phosphate (P-15 mer-15 mer), a 5’ end phosphate together with five PTs (P-15 mer-5PT-10 mer), five PTs only (15 mer-5PT-10 mer), or were not modified (15 mer-15 mer). The orange braces represent the complementary sequences. (D) Transformation efficiencies were calculated by counting the number of colonies in triplicate experiments. 1×10 8 competent cells were used for transforming the mixture of 50 ng of each purified PCR product after undergoing SLIC experiments. Single asterisk; P-values lower than 0.05, double asterisk; lower than 0.01, triple asterisks; P-value lower than 0.001. All experiments were repeated three times. (E) Representative plates of (D) are shown. CFU; colony forming unit, Ctl; control, Lambda Exo; lambda exonuclease, T5 exo; T5 exonuclease.

Article Snippet: 10 ng of pBluescript II SK+ was used as the PCR template. pX330-puro was a gift from Sandra Martha Gomes Dias (Addgene plasmid # 110403; http://n2t.net/addgene:110403 ; RRID:Addgene 110403).

Techniques: Plasmid Preparation, Modification, Transformation Assay, Purification, Control

Journal: Non-coding RNA

Article Title: A New Specific and Sensitive RT-qPCR Method Based on Splinted 5' Ligation for the Quantitative Detection of RNA Species Shorter than microRNAs.

doi: 10.3390/ncrna7030059

Figure Lengend Snippet: Figure 4. Amplification of doRNA and C-doRNA by splinted 5′ ligation RT-qPCR was specific. (A) Melting curve of the qPCR reaction products following splinted 5′ ligation RT-qPCR detection of doRNA (in dark blue) or C-doRNA (in light blue) in total RNA samples from cultured N2a cells. RFU, relative fluorescence unit. (B) Amplification of a single product upon splinted 5′ ligation RT-qPCR detection of RNA and C-doRNA. qPCR products were separated by 20% acrylamide/8 M urea gel electrophoresis and visualized by nucleic acid staining (RedSafe). (C) DNA contained in the bands excised from lanes 3 and 4 was extracted and cloned into the pBluescript II plasmid. DNA sequencing confirmed the identity of the expected amplified product (in color). UP, universal primer.

Article Snippet: The pBluescript II plasmid (Addgene, MA, USA) was linearized using EcoRV restriction enzyme (New England Biolabs, MA, USA), inducing a cut within the LacZ gene, followed by dephosphorylation of the blunt ends using Antarctic phosphatase enzyme (New England Biolabs, MA, USA) to prevent plasmid recircularization.

Techniques: Ligation, Quantitative RT-PCR, Cell Culture, Nucleic Acid Electrophoresis, Staining, Clone Assay, Plasmid Preparation, DNA Sequencing