The PHB-producing cells were spread on the agar plate containing LipidGreen1 at a final concentration of 25 µg/mL and cultured for 20 h at 37°C. Accumulation of intracellular PHB was viewed under ultraviolet light (302 nm). E. coli XL1-Blue, which contains only the pBluescript II SK+ vector (Agilent Technologies, USA), was prepared as a negative control. Subsequently, the PHB-producing and PHB-non-producing cells were scraped from the surface of the agar plates and suspended in 100 µL of phosphate-buffered saline (PBS, pH 7.2, 20 mM). Ten microliters of the suspensions were placed on slide glass and used for microscopic observation by fluorescence microscope (Nikon, Japan) with a green fluorescence filter (Green Excitation 460–500 nm, Emission 510–560 mm).
Pbluescript 2 sk vector
Manufactured by Agilent Technologies
Sourced in United States
The PBluescript II SK+ vector is a small, high-copy-number cloning vector commonly used for various molecular biology applications. It contains the pUC origin of replication, an ampicillin resistance gene, and multiple cloning sites that allow for the insertion and expression of foreign DNA sequences.
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Lab products found in correlation
Fluorescence microscope, by Nikon (1 mentions)
Tubeseq service, by Eurofins (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Sepasol rna 1 super g, by Nacalai Tesque (1 mentions)
Isostrip mouse monoclonal antibody isotyping kit, by Roche (1 mentions)
Revertra ace, by Toyobo (1 mentions)
Oligo dt primer, by Takara Bio (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Wizard sv genomic dna purification system, by Promega (1 mentions)
5 protocols using pbluescript 2 sk vector
1
Visualizing Intracellular PHB Accumulation
2
Identification of SS-like Peptide Precursors
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Two transcripts encoding precursors of SS-like peptides were identified by analysis of A. rubens neural transcriptome sequence data—ArSSP1 (GenBank: KT601708 ) and ArSSP2 (GenBank: MN257487 ). The sequence of ArSSP1 has been reported previously [48 (link)]. The sequence of ArSSP2 is reported here for the first time and was identified on account of its similarity to precursors of SS-like peptides that have been identified in other echinoderms [49 (link)]. Informed by the assembled transcript sequences of ArSSP1 and ArSSP2, cDNAs comprising the complete open reading frame of ArSSP1 and ArSSP2 were amplified from A. rubens radial nerve cord cDNA by PCR using specific primers (electronic supplementary material, table S1) and Q5 High-fidelity DNA polymerase (NEB, Hitchin, UK; cat. no. M0491S), cloned into pBluescript II SK+ vector (Agilent Technologies, Santa Clara, USA; cat. no. 212205) and then sequenced (TubeSeq service; Eurofins Genomics, Ebersberg, Germany).
3
Site-Directed Mutagenesis of CD148
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The c‐terminally hemagglutinin (HA) epitope‐tagged CD148 cDNA1 was subcloned into the XhoI/EcoRI sites in pBlueScript II SK (+) vector (Agilent Technologies, La Jolla, CA). The Q5 Site‐Directed Mutagenesis Kit (New England BioLabs, Ipswich, MA) was used to introduce the Q276P and R326Q mutations. The following PCR primers were designed to generate each mutation: Q276P (Forward: TAT CTT CTA CCA TCA AAT AAG ACA, Reverse: CGG GTT GAT GTT GTA TTG AAC CCC); R326Q (Forward: CAG CAG TCC CAA GAC ACG GAA GTC, Reverse: GCC GGA GGA TGG GTC CAC AGG TCC). The mutagenesis reactions were performed sequentially to create Q276P/R326Q mutations. The correct mutations were confirmed by DNA sequencing. There were no additional mutations in CD148 cDNA sequence. The mutated CD148 cDNA was then subcloned into the XhoI/EcoRI sites in the LZRS‐IRES‐Zeo retroviral vector44 that contains a zeocin resistant cassette. The LZRS‐IRES‐Zeo vector that expresses WT CD148 has been prepared previously.15
4
Cloning and Sequencing of Murine mAb G196
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Total RNA was extracted from 3 × 106 cells of the murine hybridoma clone G196–3 by using Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan). Total RNA was reverse transcribed into cDNA by using oligo-dT14 and ReverTra Ace (mutated M-MLV reverse transcriptase, RNaseH negative) (TOYOBO, Osaka, Japan). DNA fragments encoding the heavy and light chain were PCR amplified using the Pfu polymerase (BioAcademia). The primers used, containing the BamHI and EcoRI restriction sites, for amplifying heavy chain genes were VH1-1S and IgG2-1AS; for light chain genes the primers were VK-1S and CK-2AS (Supplementary Table 2 ). The amplified fragments were digested with BamHI and EcoRI and cloned into the pBlueScript II SK(+) vector (Agilent Technologies, Santa Clara, CA, USA) digested with the same two enzymes. The DNA sequences of the heavy and light chain genes were determined by automated sequencing using T7 and T3 sequencing primers with BigDye sequencing reagent (ThermoFisher Scientific) and an ABI 3100 automated capillary DNA sequencer (ThermoFisher Scientific).
The isotype of mAb G196 was identified as mouse IgG1 using an IsoStrip mouse monoclonal antibody isotyping kit (Roche Diagnostics, Basel, Switzerland).
The isotype of mAb G196 was identified as mouse IgG1 using an IsoStrip mouse monoclonal antibody isotyping kit (Roche Diagnostics, Basel, Switzerland).
5
Cloning and Sequencing of PTH Gene
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We synthesized PTH cDNA from RNA that was reverse transcribed with an oligo(dT) primer (TaKaRa Biotechnology), amplified a 697-bp cDNA product (coding capacity, between the 61st and 408th nucleotides) and cloned it into the pBluescript II SK(+) vector (Agilent Technologies, Santa Clara, CA). Two independent clones were sequenced on both strands. Genomic DNA was extracted from the MFH+P sample and the patients’ leukocytes using the Wizard SV Genomic DNA Purification System (Promega, Madison, WI) and a QIAamp DNA Mini kit (QIAGEN, Hilden, Germany), respectively. To sequence the promoter region, nine overlapping DNA fragments (approximately 800 bp in size), covering a region from 5.7 kbp upstream to 360 bp downstream of the transcription start site, were amplified using PCR. This region was cloned into the pBluescript II SK(+) vector, and two to four independent clones were sequenced on both strands. PTH promoter genomic DNA from the tumor was converted with bisulfite using a MethylEasy Xceed Rapid DNA Bisulphite Modification Kit (Human Genetic Signatures, Sydney, Australia). A 232-bp fragment corresponding to −1312 to −1081 bp from the transcription start site, which contains two CpG sites, was amplified by PCR and cloned into the pGEM-T Easy vector (Promega, Madison, WI). Nine clones from the tumor and three clones from the PA were sequenced on both strands.
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