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Bglii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 226 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Transduction:

Article Title: AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF) and Endonuclease G (EndoG) While Promoting Caspase Activation during Cardiac Ischemia
Article Snippet: Annealed primers have protuberant ends compatible with restriction sites for HindIII and BglII (Takara, Dalian, China), ready to be introduced directly into pSUPER.retro.puro (Oligoengine Inc., Seattle, WA, USA) previously digested by HindIII and BglII (Takara). .. The pLVTHM plasmid is the lentiviral vector used for the transduction of specific shRNAi into mammalian cells.

Clone Assay:

Article Title: VE-PTP stabilizes VE-cadherin junctions and the endothelial barrier via a phosphatase-independent mechanism
Article Snippet: CFP-tagged murine full-length VE-PTP aa 1–1,998 (WT VE-PTP) was cloned from mVE-PTP cDNA , a gift from D. Vestweber (Max Planck Institute for Molecular Biomedicine, Münster, Germany), into pAmCyan1-C1 (catalog number PT3478-5 or 632441; Clontech) at flanking 5′/3′-SalI restriction sites. .. Plum-tagged deletion mutant VE-PTP aa 1,422–1,998 was generated from CFP-tagged Δ16FN and inserted into pPlum-C1 vector at 5′-BglII and 3′-EcoRI. pPlum-C1 vector was constructed using the pAmCyan1-C1 vector as the backbone, where cDNA for the mPlum protein (catalog number 632527; Clontech) replaced cDNA for the AmCyan1 protein.

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: To generate fusion vectors, the appropriate cloning vector and an EGFP fusion vector were digested, either sequentially or doubly, with the appropriate enzymes and ligated together after gel purification. .. To prepare mTFP1 and mWasabi C-terminal fusions, the following digests were performed: human β-actin, NheI and BglII (Clontech); human α-tubulin, NheI and BglII (Clontech); human light chain clathrin, NheI and BglII (George Patterson, NIH); human lamin B1, NheI and BglII (George Patterson, NIH); human fibrillarin, AgeI and BglII (Evrogen); human vinculin, NheI and EcoRI (Open Biosystems, Huntsville, AL); peroximal targeting signal 1 (PTS1 – peroxisomes), AgeI and BspEI (Clontech); chicken protein tyrosine kinase 2, AgeI and BglII (Clare Waterman-Storer, NIH); human annexin (A4), AgeI and BspEI (Alen Piljic, EMBL, Heidelberg); human RhoB GTPase with an N-terminal c-Myc epitope tag (endosomes), AgeI and BspEI (Clontech); and the 20-amino acid farnesylation signal from c-Ha-Ras, AgeI and BspEI (membrane, Clontech).

Article Title: Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy
Article Snippet: The PCR products were gel purified, digested, and ligated to identically treated EGFP-C1 and EGFP-N1 cloning vectors. .. To construct the mEos4a/b fusions to the C-terminus of the fluorescent protein (number of linker amino acids in parenthesis), the subsequent digests were performed: human lamin A (18), NheI and BglII (cDNA source: D. Gilbert, FSU; NM_170707.2); human α-tubulin (18), NheI and BglII (cDNA source: Clontech; NM_006082); human H2B (6), BglII and NheI (histones, cDNA source: G. Patterson, NIH; NM_021058.3).

Article Title: Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein
Article Snippet: The purified and digested PCR products were ligated into similarly digested pEGFP-C1 and pEGFP-N1 cloning vectors to create pmCardinal-C1 and pmCardinal-N1. .. To prepare C-terminal fusions to mCardinal, the following digests were performed: human β-actin (NM_001101.3, Clontech), NheI and BglII; human α-tubulin (NM_006082, Clontech), NheI and BglII; human Rab4a (NM_004578.2, Viki Allen, University of Manchester, U.K.), BspEI and BamHI; human lamin B1 (NM_005573.2, George Patterson, NIH), EcoRI and BamHI; human myotilin (NM_006790.1, Origene), AgeI and BspEI; human fibrillarin (NM_001436.3, Evrogen), BglII and BamHI; human tight junction protein ZO1 (NM_003257.1, Origene), AgeI and BspEI; human VE cadherin (NM_001795.3, Origene), BglII and EcoRI; and the 20-amino-acid farnesylation signal from c-Ha-Ras (NM_001130442.1, Clontech), AgeI and BspEI.

Article Title: A naturally-monomeric infrared fluorescent protein for protein labeling in vivo
Article Snippet: The PCR products were gel purified, digested and ligated into EGFP-C1 or EGFP-N1 cloning vectors respectively, resulting in mIFP C1 and N1 cloning vectors. .. To construct the mIFP C-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human β-actin (30), NheI and BglII (cDNA source: Clontech, Mountain View, CA, USA; NM_001101.3); CAF1 (22), AgeI and BspEI (mouse chromatin assembly factor; cDNA source: A. Gunjan, Florida State University, Tallahassee, FL, USA; NM_013733.3); human light chain clathrin (27), NheI and BglII (cDNA source: G. Patterson, National Institutes of Health, Bethesda, MD, USA; NM_001834.2); human endosomes (26), NheI and BspEI (human RhoB GTPase; cDNA source: Clontech; NM_004040.2); human fibrillarin (19), AgeI and BspEI (cDNA source: Evrogen, Moscow, Russia; NM_001436.3); H2B (10), BglII and NheI (human histone 2B, cDNA source: G. Patterson, NIH; NM_021058.3); human lamin A/C (30), NheI and BglII (cDNA source: D. Gilbert, Florida State University; NM_170707.2); human lasp1 (22) NheI and BglII (cDNA source: Origene; NM_006148.3); human myotilin (26), AgeI and BspEI (cDNA source: Origene; NM_006790.2); human Rab4a (19), BglII and BamHI (cDNA source: V. Allen, University of Manchester, Manchester, UK; NM_004578.3); rat sEpsin (30) NheI and BglII (cDNA source: Origene; NM_019585.3); human α-tubulin (30), NheI and BglII (cDNA source: Clontech; NM_006082); human vinculin (35) NheI and EcoRI (cDNA source: Origene; NM_003373.3).

Transfection:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: To prepare mTFP1 and mWasabi C-terminal fusions, the following digests were performed: human β-actin, NheI and BglII (Clontech); human α-tubulin, NheI and BglII (Clontech); human light chain clathrin, NheI and BglII (George Patterson, NIH); human lamin B1, NheI and BglII (George Patterson, NIH); human fibrillarin, AgeI and BglII (Evrogen); human vinculin, NheI and EcoRI (Open Biosystems, Huntsville, AL); peroximal targeting signal 1 (PTS1 – peroxisomes), AgeI and BspEI (Clontech); chicken protein tyrosine kinase 2, AgeI and BglII (Clare Waterman-Storer, NIH); human annexin (A4), AgeI and BspEI (Alen Piljic, EMBL, Heidelberg); human RhoB GTPase with an N-terminal c-Myc epitope tag (endosomes), AgeI and BspEI (Clontech); and the 20-amino acid farnesylation signal from c-Ha-Ras, AgeI and BspEI (membrane, Clontech). .. DNA for mammalian transfection was prepared by either the Plasmid Midi or Maxi kit (QIAGEN).

Article Title: A naturally-monomeric infrared fluorescent protein for protein labeling in vivo
Article Snippet: To construct the mIFP C-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human β-actin (30), NheI and BglII (cDNA source: Clontech, Mountain View, CA, USA; NM_001101.3); CAF1 (22), AgeI and BspEI (mouse chromatin assembly factor; cDNA source: A. Gunjan, Florida State University, Tallahassee, FL, USA; NM_013733.3); human light chain clathrin (27), NheI and BglII (cDNA source: G. Patterson, National Institutes of Health, Bethesda, MD, USA; NM_001834.2); human endosomes (26), NheI and BspEI (human RhoB GTPase; cDNA source: Clontech; NM_004040.2); human fibrillarin (19), AgeI and BspEI (cDNA source: Evrogen, Moscow, Russia; NM_001436.3); H2B (10), BglII and NheI (human histone 2B, cDNA source: G. Patterson, NIH; NM_021058.3); human lamin A/C (30), NheI and BglII (cDNA source: D. Gilbert, Florida State University; NM_170707.2); human lasp1 (22) NheI and BglII (cDNA source: Origene; NM_006148.3); human myotilin (26), AgeI and BspEI (cDNA source: Origene; NM_006790.2); human Rab4a (19), BglII and BamHI (cDNA source: V. Allen, University of Manchester, Manchester, UK; NM_004578.3); rat sEpsin (30) NheI and BglII (cDNA source: Origene; NM_019585.3); human α-tubulin (30), NheI and BglII (cDNA source: Clontech; NM_006082); human vinculin (35) NheI and EcoRI (cDNA source: Origene; NM_003373.3). .. DNA for transfection was prepared using the Plasmid Maxi kit (Qiagen).

Amplification:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: The FPs were amplified with a 5' primer encoding an AgeI site and a 3' primer encoding either a BspEI (C1) or Not1 (N1) site. .. To prepare mTFP1 and mWasabi C-terminal fusions, the following digests were performed: human β-actin, NheI and BglII (Clontech); human α-tubulin, NheI and BglII (Clontech); human light chain clathrin, NheI and BglII (George Patterson, NIH); human lamin B1, NheI and BglII (George Patterson, NIH); human fibrillarin, AgeI and BglII (Evrogen); human vinculin, NheI and EcoRI (Open Biosystems, Huntsville, AL); peroximal targeting signal 1 (PTS1 – peroxisomes), AgeI and BspEI (Clontech); chicken protein tyrosine kinase 2, AgeI and BglII (Clare Waterman-Storer, NIH); human annexin (A4), AgeI and BspEI (Alen Piljic, EMBL, Heidelberg); human RhoB GTPase with an N-terminal c-Myc epitope tag (endosomes), AgeI and BspEI (Clontech); and the 20-amino acid farnesylation signal from c-Ha-Ras, AgeI and BspEI (membrane, Clontech).

Article Title: Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy
Article Snippet: Utilizing PCR the mEos4a and mEos4b cDNAs were amplified with a 5′ primer encoding an AgeI site and a 3′ primer containing either a BspEI (C1) or NotI (N1) site for creating the C-terminal and N-terminal (with respect to the FP) cloning vectors. .. To construct the mEos4a/b fusions to the C-terminus of the fluorescent protein (number of linker amino acids in parenthesis), the subsequent digests were performed: human lamin A (18), NheI and BglII (cDNA source: D. Gilbert, FSU; NM_170707.2); human α-tubulin (18), NheI and BglII (cDNA source: Clontech; NM_006082); human H2B (6), BglII and NheI (histones, cDNA source: G. Patterson, NIH; NM_021058.3).

Article Title: Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein
Article Snippet: A cDNA fragment encoding each protein domain was PCR amplified with primers containing the appropriate restriction enzyme sites and ligated into pmCardinal-C1 or pmCardinal-N1. .. To prepare C-terminal fusions to mCardinal, the following digests were performed: human β-actin (NM_001101.3, Clontech), NheI and BglII; human α-tubulin (NM_006082, Clontech), NheI and BglII; human Rab4a (NM_004578.2, Viki Allen, University of Manchester, U.K.), BspEI and BamHI; human lamin B1 (NM_005573.2, George Patterson, NIH), EcoRI and BamHI; human myotilin (NM_006790.1, Origene), AgeI and BspEI; human fibrillarin (NM_001436.3, Evrogen), BglII and BamHI; human tight junction protein ZO1 (NM_003257.1, Origene), AgeI and BspEI; human VE cadherin (NM_001795.3, Origene), BglII and EcoRI; and the 20-amino-acid farnesylation signal from c-Ha-Ras (NM_001130442.1, Clontech), AgeI and BspEI.

Article Title: A naturally-monomeric infrared fluorescent protein for protein labeling in vivo
Article Snippet: The mIFP cDNA was PCR amplified with a 5’ primer encoding an AgeI site and a 3’ primer encoding either a BspEI (C1) or NotI (N1) site, in reference to mIFP. .. To construct the mIFP C-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human β-actin (30), NheI and BglII (cDNA source: Clontech, Mountain View, CA, USA; NM_001101.3); CAF1 (22), AgeI and BspEI (mouse chromatin assembly factor; cDNA source: A. Gunjan, Florida State University, Tallahassee, FL, USA; NM_013733.3); human light chain clathrin (27), NheI and BglII (cDNA source: G. Patterson, National Institutes of Health, Bethesda, MD, USA; NM_001834.2); human endosomes (26), NheI and BspEI (human RhoB GTPase; cDNA source: Clontech; NM_004040.2); human fibrillarin (19), AgeI and BspEI (cDNA source: Evrogen, Moscow, Russia; NM_001436.3); H2B (10), BglII and NheI (human histone 2B, cDNA source: G. Patterson, NIH; NM_021058.3); human lamin A/C (30), NheI and BglII (cDNA source: D. Gilbert, Florida State University; NM_170707.2); human lasp1 (22) NheI and BglII (cDNA source: Origene; NM_006148.3); human myotilin (26), AgeI and BspEI (cDNA source: Origene; NM_006790.2); human Rab4a (19), BglII and BamHI (cDNA source: V. Allen, University of Manchester, Manchester, UK; NM_004578.3); rat sEpsin (30) NheI and BglII (cDNA source: Origene; NM_019585.3); human α-tubulin (30), NheI and BglII (cDNA source: Clontech; NM_006082); human vinculin (35) NheI and EcoRI (cDNA source: Origene; NM_003373.3).

Polymerase Chain Reaction:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: The purified and digested PCR products were ligated into similarly digested EGFP-C1 and EGFP-N1 cloning vector backbones. .. To prepare mTFP1 and mWasabi C-terminal fusions, the following digests were performed: human β-actin, NheI and BglII (Clontech); human α-tubulin, NheI and BglII (Clontech); human light chain clathrin, NheI and BglII (George Patterson, NIH); human lamin B1, NheI and BglII (George Patterson, NIH); human fibrillarin, AgeI and BglII (Evrogen); human vinculin, NheI and EcoRI (Open Biosystems, Huntsville, AL); peroximal targeting signal 1 (PTS1 – peroxisomes), AgeI and BspEI (Clontech); chicken protein tyrosine kinase 2, AgeI and BglII (Clare Waterman-Storer, NIH); human annexin (A4), AgeI and BspEI (Alen Piljic, EMBL, Heidelberg); human RhoB GTPase with an N-terminal c-Myc epitope tag (endosomes), AgeI and BspEI (Clontech); and the 20-amino acid farnesylation signal from c-Ha-Ras, AgeI and BspEI (membrane, Clontech).

Article Title: Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy
Article Snippet: The PCR products were gel purified, digested, and ligated to identically treated EGFP-C1 and EGFP-N1 cloning vectors. .. To construct the mEos4a/b fusions to the C-terminus of the fluorescent protein (number of linker amino acids in parenthesis), the subsequent digests were performed: human lamin A (18), NheI and BglII (cDNA source: D. Gilbert, FSU; NM_170707.2); human α-tubulin (18), NheI and BglII (cDNA source: Clontech; NM_006082); human H2B (6), BglII and NheI (histones, cDNA source: G. Patterson, NIH; NM_021058.3).

Article Title: Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein
Article Snippet: A cDNA fragment encoding each protein domain was PCR amplified with primers containing the appropriate restriction enzyme sites and ligated into pmCardinal-C1 or pmCardinal-N1. .. To prepare C-terminal fusions to mCardinal, the following digests were performed: human β-actin (NM_001101.3, Clontech), NheI and BglII; human α-tubulin (NM_006082, Clontech), NheI and BglII; human Rab4a (NM_004578.2, Viki Allen, University of Manchester, U.K.), BspEI and BamHI; human lamin B1 (NM_005573.2, George Patterson, NIH), EcoRI and BamHI; human myotilin (NM_006790.1, Origene), AgeI and BspEI; human fibrillarin (NM_001436.3, Evrogen), BglII and BamHI; human tight junction protein ZO1 (NM_003257.1, Origene), AgeI and BspEI; human VE cadherin (NM_001795.3, Origene), BglII and EcoRI; and the 20-amino-acid farnesylation signal from c-Ha-Ras (NM_001130442.1, Clontech), AgeI and BspEI.

Article Title: A naturally-monomeric infrared fluorescent protein for protein labeling in vivo
Article Snippet: The PCR products were gel purified, digested and ligated into EGFP-C1 or EGFP-N1 cloning vectors respectively, resulting in mIFP C1 and N1 cloning vectors. .. To construct the mIFP C-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human β-actin (30), NheI and BglII (cDNA source: Clontech, Mountain View, CA, USA; NM_001101.3); CAF1 (22), AgeI and BspEI (mouse chromatin assembly factor; cDNA source: A. Gunjan, Florida State University, Tallahassee, FL, USA; NM_013733.3); human light chain clathrin (27), NheI and BglII (cDNA source: G. Patterson, National Institutes of Health, Bethesda, MD, USA; NM_001834.2); human endosomes (26), NheI and BspEI (human RhoB GTPase; cDNA source: Clontech; NM_004040.2); human fibrillarin (19), AgeI and BspEI (cDNA source: Evrogen, Moscow, Russia; NM_001436.3); H2B (10), BglII and NheI (human histone 2B, cDNA source: G. Patterson, NIH; NM_021058.3); human lamin A/C (30), NheI and BglII (cDNA source: D. Gilbert, Florida State University; NM_170707.2); human lasp1 (22) NheI and BglII (cDNA source: Origene; NM_006148.3); human myotilin (26), AgeI and BspEI (cDNA source: Origene; NM_006790.2); human Rab4a (19), BglII and BamHI (cDNA source: V. Allen, University of Manchester, Manchester, UK; NM_004578.3); rat sEpsin (30) NheI and BglII (cDNA source: Origene; NM_019585.3); human α-tubulin (30), NheI and BglII (cDNA source: Clontech; NM_006082); human vinculin (35) NheI and EcoRI (cDNA source: Origene; NM_003373.3).

Mutagenesis:

Article Title: VE-PTP stabilizes VE-cadherin junctions and the endothelial barrier via a phosphatase-independent mechanism
Article Snippet: .. Plum-tagged deletion mutant VE-PTP aa 1,422–1,998 was generated from CFP-tagged Δ16FN and inserted into pPlum-C1 vector at 5′-BglII and 3′-EcoRI. pPlum-C1 vector was constructed using the pAmCyan1-C1 vector as the backbone, where cDNA for the mPlum protein (catalog number 632527; Clontech) replaced cDNA for the AmCyan1 protein. .. CFP-tagged VE-PTP PI mutant with a D/A mutation at aa 1,871 was generated from WT VE-PTP using site-directed mutagenesis.

Subcloning:

Article Title: AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF) and Endonuclease G (EndoG) While Promoting Caspase Activation during Cardiac Ischemia
Article Snippet: The subcloning strategy is based on choosing a sequence into the target gene, which is 19 nucleotides long. .. Annealed primers have protuberant ends compatible with restriction sites for HindIII and BglII (Takara, Dalian, China), ready to be introduced directly into pSUPER.retro.puro (Oligoengine Inc., Seattle, WA, USA) previously digested by HindIII and BglII (Takara).

Generated:

Article Title: VE-PTP stabilizes VE-cadherin junctions and the endothelial barrier via a phosphatase-independent mechanism
Article Snippet: .. Plum-tagged deletion mutant VE-PTP aa 1,422–1,998 was generated from CFP-tagged Δ16FN and inserted into pPlum-C1 vector at 5′-BglII and 3′-EcoRI. pPlum-C1 vector was constructed using the pAmCyan1-C1 vector as the backbone, where cDNA for the mPlum protein (catalog number 632527; Clontech) replaced cDNA for the AmCyan1 protein. .. CFP-tagged VE-PTP PI mutant with a D/A mutation at aa 1,871 was generated from WT VE-PTP using site-directed mutagenesis.

Construct:

Article Title: VE-PTP stabilizes VE-cadherin junctions and the endothelial barrier via a phosphatase-independent mechanism
Article Snippet: .. Plum-tagged deletion mutant VE-PTP aa 1,422–1,998 was generated from CFP-tagged Δ16FN and inserted into pPlum-C1 vector at 5′-BglII and 3′-EcoRI. pPlum-C1 vector was constructed using the pAmCyan1-C1 vector as the backbone, where cDNA for the mPlum protein (catalog number 632527; Clontech) replaced cDNA for the AmCyan1 protein. .. CFP-tagged VE-PTP PI mutant with a D/A mutation at aa 1,871 was generated from WT VE-PTP using site-directed mutagenesis.

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: All of the other mTFP1 and mWasabi vectors were constructed using C1 and N1 (Clontech-style) cloning vectors. .. To prepare mTFP1 and mWasabi C-terminal fusions, the following digests were performed: human β-actin, NheI and BglII (Clontech); human α-tubulin, NheI and BglII (Clontech); human light chain clathrin, NheI and BglII (George Patterson, NIH); human lamin B1, NheI and BglII (George Patterson, NIH); human fibrillarin, AgeI and BglII (Evrogen); human vinculin, NheI and EcoRI (Open Biosystems, Huntsville, AL); peroximal targeting signal 1 (PTS1 – peroxisomes), AgeI and BspEI (Clontech); chicken protein tyrosine kinase 2, AgeI and BglII (Clare Waterman-Storer, NIH); human annexin (A4), AgeI and BspEI (Alen Piljic, EMBL, Heidelberg); human RhoB GTPase with an N-terminal c-Myc epitope tag (endosomes), AgeI and BspEI (Clontech); and the 20-amino acid farnesylation signal from c-Ha-Ras, AgeI and BspEI (membrane, Clontech).

Article Title: Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy
Article Snippet: .. To construct the mEos4a/b fusions to the C-terminus of the fluorescent protein (number of linker amino acids in parenthesis), the subsequent digests were performed: human lamin A (18), NheI and BglII (cDNA source: D. Gilbert, FSU; NM_170707.2); human α-tubulin (18), NheI and BglII (cDNA source: Clontech; NM_006082); human H2B (6), BglII and NheI (histones, cDNA source: G. Patterson, NIH; NM_021058.3). ..

Article Title: AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF) and Endonuclease G (EndoG) While Promoting Caspase Activation during Cardiac Ischemia
Article Snippet: 4.4. shRNA Knockdown of AKT2, Endo G and AIF Gene Silencing—In order to obtain the rat-specific small interfering RNA (siRNA) construct, we designed several primer pairs against the target gene; the selected sequences are shown in . .. Annealed primers have protuberant ends compatible with restriction sites for HindIII and BglII (Takara, Dalian, China), ready to be introduced directly into pSUPER.retro.puro (Oligoengine Inc., Seattle, WA, USA) previously digested by HindIII and BglII (Takara).

Article Title: A naturally-monomeric infrared fluorescent protein for protein labeling in vivo
Article Snippet: .. To construct the mIFP C-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human β-actin (30), NheI and BglII (cDNA source: Clontech, Mountain View, CA, USA; NM_001101.3); CAF1 (22), AgeI and BspEI (mouse chromatin assembly factor; cDNA source: A. Gunjan, Florida State University, Tallahassee, FL, USA; NM_013733.3); human light chain clathrin (27), NheI and BglII (cDNA source: G. Patterson, National Institutes of Health, Bethesda, MD, USA; NM_001834.2); human endosomes (26), NheI and BspEI (human RhoB GTPase; cDNA source: Clontech; NM_004040.2); human fibrillarin (19), AgeI and BspEI (cDNA source: Evrogen, Moscow, Russia; NM_001436.3); H2B (10), BglII and NheI (human histone 2B, cDNA source: G. Patterson, NIH; NM_021058.3); human lamin A/C (30), NheI and BglII (cDNA source: D. Gilbert, Florida State University; NM_170707.2); human lasp1 (22) NheI and BglII (cDNA source: Origene; NM_006148.3); human myotilin (26), AgeI and BspEI (cDNA source: Origene; NM_006790.2); human Rab4a (19), BglII and BamHI (cDNA source: V. Allen, University of Manchester, Manchester, UK; NM_004578.3); rat sEpsin (30) NheI and BglII (cDNA source: Origene; NM_019585.3); human α-tubulin (30), NheI and BglII (cDNA source: Clontech; NM_006082); human vinculin (35) NheI and EcoRI (cDNA source: Origene; NM_003373.3). .. To prepare the mIFP N-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human calnexin (14), AgeI and NotI (cDNA source: Origene; NM_001746.3); human CENP-B (22), BamHI and NotI (A. Khodjakov, Wadsworth Center, Albany, NY, USA; NM_001810.5); Cx43 (7), BamHI and NotI (rat α-1 connexin 43 cDNA source: M. Falk, Lehigh University, Bethlehem, PA, USA; NM_001004099.1); human EB3 (7), BglII and BamHI (cDNA source: L. Cassimeris, Lehigh University; NM_012326.2); H1 (10), BamHI and NotI (mouse histone 1, cDNA source: G. Patterson, NIH; NM_008197.3); H2B (6), BamHI and NotI (human histone 2B, cDNA source: G. Patterson, NIH; NM_021058.3); human keratin18 (17), EcoRI and NotI (cDNA source: Open Biosystems, Huntsville, AL, USA; NM_199187.1); rat lysosomal membrane glycoprotein 1 (20), BamHI and NotI (LAMP1; cDNA source: G. Patterson, NIH; NM_012857.1); lifeact (7), BamHI and NotI (cDNA source: Integrated DNA Technologies, Coralville, IA, USA); human MAPTau (10), AgeI and NotI (cDNA source: Origene; NM_016841.4); human nucleoporin 50 kDa (10), BamHI and NotI (NUP50; cDNA source: Origene; NM_007172.3); human peroxisomal membrane protein (10), NotI and AgeI (PMP; cDNA source: Origene; NM_018663.1); human translocase outer mitochondria membrane 20 (10), (TOMM-20; cDNA source: Origene; NM_014765.2); human zyxin (6), BamHI and NotI (cDNA source: Origene; NM_003461.4).

Purification:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: The purified and digested PCR products were ligated into similarly digested EGFP-C1 and EGFP-N1 cloning vector backbones. .. To prepare mTFP1 and mWasabi C-terminal fusions, the following digests were performed: human β-actin, NheI and BglII (Clontech); human α-tubulin, NheI and BglII (Clontech); human light chain clathrin, NheI and BglII (George Patterson, NIH); human lamin B1, NheI and BglII (George Patterson, NIH); human fibrillarin, AgeI and BglII (Evrogen); human vinculin, NheI and EcoRI (Open Biosystems, Huntsville, AL); peroximal targeting signal 1 (PTS1 – peroxisomes), AgeI and BspEI (Clontech); chicken protein tyrosine kinase 2, AgeI and BglII (Clare Waterman-Storer, NIH); human annexin (A4), AgeI and BspEI (Alen Piljic, EMBL, Heidelberg); human RhoB GTPase with an N-terminal c-Myc epitope tag (endosomes), AgeI and BspEI (Clontech); and the 20-amino acid farnesylation signal from c-Ha-Ras, AgeI and BspEI (membrane, Clontech).

Article Title: Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy
Article Snippet: The PCR products were gel purified, digested, and ligated to identically treated EGFP-C1 and EGFP-N1 cloning vectors. .. To construct the mEos4a/b fusions to the C-terminus of the fluorescent protein (number of linker amino acids in parenthesis), the subsequent digests were performed: human lamin A (18), NheI and BglII (cDNA source: D. Gilbert, FSU; NM_170707.2); human α-tubulin (18), NheI and BglII (cDNA source: Clontech; NM_006082); human H2B (6), BglII and NheI (histones, cDNA source: G. Patterson, NIH; NM_021058.3).

Article Title: Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein
Article Snippet: The purified and digested PCR products were ligated into similarly digested pEGFP-C1 and pEGFP-N1 cloning vectors to create pmCardinal-C1 and pmCardinal-N1. .. To prepare C-terminal fusions to mCardinal, the following digests were performed: human β-actin (NM_001101.3, Clontech), NheI and BglII; human α-tubulin (NM_006082, Clontech), NheI and BglII; human Rab4a (NM_004578.2, Viki Allen, University of Manchester, U.K.), BspEI and BamHI; human lamin B1 (NM_005573.2, George Patterson, NIH), EcoRI and BamHI; human myotilin (NM_006790.1, Origene), AgeI and BspEI; human fibrillarin (NM_001436.3, Evrogen), BglII and BamHI; human tight junction protein ZO1 (NM_003257.1, Origene), AgeI and BspEI; human VE cadherin (NM_001795.3, Origene), BglII and EcoRI; and the 20-amino-acid farnesylation signal from c-Ha-Ras (NM_001130442.1, Clontech), AgeI and BspEI.

Article Title: A naturally-monomeric infrared fluorescent protein for protein labeling in vivo
Article Snippet: The PCR products were gel purified, digested and ligated into EGFP-C1 or EGFP-N1 cloning vectors respectively, resulting in mIFP C1 and N1 cloning vectors. .. To construct the mIFP C-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human β-actin (30), NheI and BglII (cDNA source: Clontech, Mountain View, CA, USA; NM_001101.3); CAF1 (22), AgeI and BspEI (mouse chromatin assembly factor; cDNA source: A. Gunjan, Florida State University, Tallahassee, FL, USA; NM_013733.3); human light chain clathrin (27), NheI and BglII (cDNA source: G. Patterson, National Institutes of Health, Bethesda, MD, USA; NM_001834.2); human endosomes (26), NheI and BspEI (human RhoB GTPase; cDNA source: Clontech; NM_004040.2); human fibrillarin (19), AgeI and BspEI (cDNA source: Evrogen, Moscow, Russia; NM_001436.3); H2B (10), BglII and NheI (human histone 2B, cDNA source: G. Patterson, NIH; NM_021058.3); human lamin A/C (30), NheI and BglII (cDNA source: D. Gilbert, Florida State University; NM_170707.2); human lasp1 (22) NheI and BglII (cDNA source: Origene; NM_006148.3); human myotilin (26), AgeI and BspEI (cDNA source: Origene; NM_006790.2); human Rab4a (19), BglII and BamHI (cDNA source: V. Allen, University of Manchester, Manchester, UK; NM_004578.3); rat sEpsin (30) NheI and BglII (cDNA source: Origene; NM_019585.3); human α-tubulin (30), NheI and BglII (cDNA source: Clontech; NM_006082); human vinculin (35) NheI and EcoRI (cDNA source: Origene; NM_003373.3).

Sequencing:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech). .. To prepare mTFP1 and mWasabi C-terminal fusions, the following digests were performed: human β-actin, NheI and BglII (Clontech); human α-tubulin, NheI and BglII (Clontech); human light chain clathrin, NheI and BglII (George Patterson, NIH); human lamin B1, NheI and BglII (George Patterson, NIH); human fibrillarin, AgeI and BglII (Evrogen); human vinculin, NheI and EcoRI (Open Biosystems, Huntsville, AL); peroximal targeting signal 1 (PTS1 – peroxisomes), AgeI and BspEI (Clontech); chicken protein tyrosine kinase 2, AgeI and BglII (Clare Waterman-Storer, NIH); human annexin (A4), AgeI and BspEI (Alen Piljic, EMBL, Heidelberg); human RhoB GTPase with an N-terminal c-Myc epitope tag (endosomes), AgeI and BspEI (Clontech); and the 20-amino acid farnesylation signal from c-Ha-Ras, AgeI and BspEI (membrane, Clontech).

Article Title: AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF) and Endonuclease G (EndoG) While Promoting Caspase Activation during Cardiac Ischemia
Article Snippet: The subcloning strategy is based on choosing a sequence into the target gene, which is 19 nucleotides long. .. Annealed primers have protuberant ends compatible with restriction sites for HindIII and BglII (Takara, Dalian, China), ready to be introduced directly into pSUPER.retro.puro (Oligoengine Inc., Seattle, WA, USA) previously digested by HindIII and BglII (Takara).

Concentration Assay:

Article Title: AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF) and Endonuclease G (EndoG) While Promoting Caspase Activation during Cardiac Ischemia
Article Snippet: Primer annealing was done in 50 mM Hepes pH 7.4, 100 mM NaCl buffer, using 1 μL of each primer stock (3 mg/mL) to a final volume of 50 μL (primer final concentration 60 μg/m). .. Annealed primers have protuberant ends compatible with restriction sites for HindIII and BglII (Takara, Dalian, China), ready to be introduced directly into pSUPER.retro.puro (Oligoengine Inc., Seattle, WA, USA) previously digested by HindIII and BglII (Takara).

Small Interfering RNA:

Article Title: AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF) and Endonuclease G (EndoG) While Promoting Caspase Activation during Cardiac Ischemia
Article Snippet: 4.4. shRNA Knockdown of AKT2, Endo G and AIF Gene Silencing—In order to obtain the rat-specific small interfering RNA (siRNA) construct, we designed several primer pairs against the target gene; the selected sequences are shown in . .. Annealed primers have protuberant ends compatible with restriction sites for HindIII and BglII (Takara, Dalian, China), ready to be introduced directly into pSUPER.retro.puro (Oligoengine Inc., Seattle, WA, USA) previously digested by HindIII and BglII (Takara).

shRNA:

Article Title: AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF) and Endonuclease G (EndoG) While Promoting Caspase Activation during Cardiac Ischemia
Article Snippet: Paragraph title: 4.4. shRNA Knockdown of AKT2, Endo G and AIF ... Annealed primers have protuberant ends compatible with restriction sites for HindIII and BglII (Takara, Dalian, China), ready to be introduced directly into pSUPER.retro.puro (Oligoengine Inc., Seattle, WA, USA) previously digested by HindIII and BglII (Takara).

Cell Culture:

Article Title: Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy
Article Snippet: Paragraph title: Construction of mammalian expression plasmids and cell culture ... To construct the mEos4a/b fusions to the C-terminus of the fluorescent protein (number of linker amino acids in parenthesis), the subsequent digests were performed: human lamin A (18), NheI and BglII (cDNA source: D. Gilbert, FSU; NM_170707.2); human α-tubulin (18), NheI and BglII (cDNA source: Clontech; NM_006082); human H2B (6), BglII and NheI (histones, cDNA source: G. Patterson, NIH; NM_021058.3).

Expressing:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: Paragraph title: Mammalian expression vectors ... To prepare mTFP1 and mWasabi C-terminal fusions, the following digests were performed: human β-actin, NheI and BglII (Clontech); human α-tubulin, NheI and BglII (Clontech); human light chain clathrin, NheI and BglII (George Patterson, NIH); human lamin B1, NheI and BglII (George Patterson, NIH); human fibrillarin, AgeI and BglII (Evrogen); human vinculin, NheI and EcoRI (Open Biosystems, Huntsville, AL); peroximal targeting signal 1 (PTS1 – peroxisomes), AgeI and BspEI (Clontech); chicken protein tyrosine kinase 2, AgeI and BglII (Clare Waterman-Storer, NIH); human annexin (A4), AgeI and BspEI (Alen Piljic, EMBL, Heidelberg); human RhoB GTPase with an N-terminal c-Myc epitope tag (endosomes), AgeI and BspEI (Clontech); and the 20-amino acid farnesylation signal from c-Ha-Ras, AgeI and BspEI (membrane, Clontech).

Article Title: Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy
Article Snippet: Paragraph title: Construction of mammalian expression plasmids and cell culture ... To construct the mEos4a/b fusions to the C-terminus of the fluorescent protein (number of linker amino acids in parenthesis), the subsequent digests were performed: human lamin A (18), NheI and BglII (cDNA source: D. Gilbert, FSU; NM_170707.2); human α-tubulin (18), NheI and BglII (cDNA source: Clontech; NM_006082); human H2B (6), BglII and NheI (histones, cDNA source: G. Patterson, NIH; NM_021058.3).

Article Title: AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF) and Endonuclease G (EndoG) While Promoting Caspase Activation during Cardiac Ischemia
Article Snippet: Annealed primers have protuberant ends compatible with restriction sites for HindIII and BglII (Takara, Dalian, China), ready to be introduced directly into pSUPER.retro.puro (Oligoengine Inc., Seattle, WA, USA) previously digested by HindIII and BglII (Takara). .. Finally, we obtained shRNA constructions into the lentiviral vector and proceeded with the employment of the RNA interference technique to silence the expression of target genes in cardiomyocytes.

Article Title: A naturally-monomeric infrared fluorescent protein for protein labeling in vivo
Article Snippet: Fusion plasmid construction The monomeric infrared fluorescent protein (mIFP) mammalian expression vectors were constructed from C1 or N1 cloning vectors (Clontech-style). .. To construct the mIFP C-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human β-actin (30), NheI and BglII (cDNA source: Clontech, Mountain View, CA, USA; NM_001101.3); CAF1 (22), AgeI and BspEI (mouse chromatin assembly factor; cDNA source: A. Gunjan, Florida State University, Tallahassee, FL, USA; NM_013733.3); human light chain clathrin (27), NheI and BglII (cDNA source: G. Patterson, National Institutes of Health, Bethesda, MD, USA; NM_001834.2); human endosomes (26), NheI and BspEI (human RhoB GTPase; cDNA source: Clontech; NM_004040.2); human fibrillarin (19), AgeI and BspEI (cDNA source: Evrogen, Moscow, Russia; NM_001436.3); H2B (10), BglII and NheI (human histone 2B, cDNA source: G. Patterson, NIH; NM_021058.3); human lamin A/C (30), NheI and BglII (cDNA source: D. Gilbert, Florida State University; NM_170707.2); human lasp1 (22) NheI and BglII (cDNA source: Origene; NM_006148.3); human myotilin (26), AgeI and BspEI (cDNA source: Origene; NM_006790.2); human Rab4a (19), BglII and BamHI (cDNA source: V. Allen, University of Manchester, Manchester, UK; NM_004578.3); rat sEpsin (30) NheI and BglII (cDNA source: Origene; NM_019585.3); human α-tubulin (30), NheI and BglII (cDNA source: Clontech; NM_006082); human vinculin (35) NheI and EcoRI (cDNA source: Origene; NM_003373.3).

IA:

Article Title: A naturally-monomeric infrared fluorescent protein for protein labeling in vivo
Article Snippet: To construct the mIFP C-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human β-actin (30), NheI and BglII (cDNA source: Clontech, Mountain View, CA, USA; NM_001101.3); CAF1 (22), AgeI and BspEI (mouse chromatin assembly factor; cDNA source: A. Gunjan, Florida State University, Tallahassee, FL, USA; NM_013733.3); human light chain clathrin (27), NheI and BglII (cDNA source: G. Patterson, National Institutes of Health, Bethesda, MD, USA; NM_001834.2); human endosomes (26), NheI and BspEI (human RhoB GTPase; cDNA source: Clontech; NM_004040.2); human fibrillarin (19), AgeI and BspEI (cDNA source: Evrogen, Moscow, Russia; NM_001436.3); H2B (10), BglII and NheI (human histone 2B, cDNA source: G. Patterson, NIH; NM_021058.3); human lamin A/C (30), NheI and BglII (cDNA source: D. Gilbert, Florida State University; NM_170707.2); human lasp1 (22) NheI and BglII (cDNA source: Origene; NM_006148.3); human myotilin (26), AgeI and BspEI (cDNA source: Origene; NM_006790.2); human Rab4a (19), BglII and BamHI (cDNA source: V. Allen, University of Manchester, Manchester, UK; NM_004578.3); rat sEpsin (30) NheI and BglII (cDNA source: Origene; NM_019585.3); human α-tubulin (30), NheI and BglII (cDNA source: Clontech; NM_006082); human vinculin (35) NheI and EcoRI (cDNA source: Origene; NM_003373.3). .. To prepare the mIFP N-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human calnexin (14), AgeI and NotI (cDNA source: Origene; NM_001746.3); human CENP-B (22), BamHI and NotI (A. Khodjakov, Wadsworth Center, Albany, NY, USA; NM_001810.5); Cx43 (7), BamHI and NotI (rat α-1 connexin 43 cDNA source: M. Falk, Lehigh University, Bethlehem, PA, USA; NM_001004099.1); human EB3 (7), BglII and BamHI (cDNA source: L. Cassimeris, Lehigh University; NM_012326.2); H1 (10), BamHI and NotI (mouse histone 1, cDNA source: G. Patterson, NIH; NM_008197.3); H2B (6), BamHI and NotI (human histone 2B, cDNA source: G. Patterson, NIH; NM_021058.3); human keratin18 (17), EcoRI and NotI (cDNA source: Open Biosystems, Huntsville, AL, USA; NM_199187.1); rat lysosomal membrane glycoprotein 1 (20), BamHI and NotI (LAMP1; cDNA source: G. Patterson, NIH; NM_012857.1); lifeact (7), BamHI and NotI (cDNA source: Integrated DNA Technologies, Coralville, IA, USA); human MAPTau (10), AgeI and NotI (cDNA source: Origene; NM_016841.4); human nucleoporin 50 kDa (10), BamHI and NotI (NUP50; cDNA source: Origene; NM_007172.3); human peroxisomal membrane protein (10), NotI and AgeI (PMP; cDNA source: Origene; NM_018663.1); human translocase outer mitochondria membrane 20 (10), (TOMM-20; cDNA source: Origene; NM_014765.2); human zyxin (6), BamHI and NotI (cDNA source: Origene; NM_003461.4).

Plasmid Preparation:

Article Title: VE-PTP stabilizes VE-cadherin junctions and the endothelial barrier via a phosphatase-independent mechanism
Article Snippet: .. Plum-tagged deletion mutant VE-PTP aa 1,422–1,998 was generated from CFP-tagged Δ16FN and inserted into pPlum-C1 vector at 5′-BglII and 3′-EcoRI. pPlum-C1 vector was constructed using the pAmCyan1-C1 vector as the backbone, where cDNA for the mPlum protein (catalog number 632527; Clontech) replaced cDNA for the AmCyan1 protein. .. CFP-tagged VE-PTP PI mutant with a D/A mutation at aa 1,871 was generated from WT VE-PTP using site-directed mutagenesis.

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech). .. To prepare mTFP1 and mWasabi C-terminal fusions, the following digests were performed: human β-actin, NheI and BglII (Clontech); human α-tubulin, NheI and BglII (Clontech); human light chain clathrin, NheI and BglII (George Patterson, NIH); human lamin B1, NheI and BglII (George Patterson, NIH); human fibrillarin, AgeI and BglII (Evrogen); human vinculin, NheI and EcoRI (Open Biosystems, Huntsville, AL); peroximal targeting signal 1 (PTS1 – peroxisomes), AgeI and BspEI (Clontech); chicken protein tyrosine kinase 2, AgeI and BglII (Clare Waterman-Storer, NIH); human annexin (A4), AgeI and BspEI (Alen Piljic, EMBL, Heidelberg); human RhoB GTPase with an N-terminal c-Myc epitope tag (endosomes), AgeI and BspEI (Clontech); and the 20-amino acid farnesylation signal from c-Ha-Ras, AgeI and BspEI (membrane, Clontech).

Article Title: Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy
Article Snippet: To construct the mEos4a/b fusions to the C-terminus of the fluorescent protein (number of linker amino acids in parenthesis), the subsequent digests were performed: human lamin A (18), NheI and BglII (cDNA source: D. Gilbert, FSU; NM_170707.2); human α-tubulin (18), NheI and BglII (cDNA source: Clontech; NM_006082); human H2B (6), BglII and NheI (histones, cDNA source: G. Patterson, NIH; NM_021058.3). .. Following digestion with the appropriate restriction enzymes and gel purification, the plasmids were ligated together with the similarly digested and gel-purified mEos4a or mEos4b cloning vector and purified using a Plasmid Maxiprep kit (Qiagen).

Article Title: AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF) and Endonuclease G (EndoG) While Promoting Caspase Activation during Cardiac Ischemia
Article Snippet: Annealed primers have protuberant ends compatible with restriction sites for HindIII and BglII (Takara, Dalian, China), ready to be introduced directly into pSUPER.retro.puro (Oligoengine Inc., Seattle, WA, USA) previously digested by HindIII and BglII (Takara). .. The pLVTHM plasmid is the lentiviral vector used for the transduction of specific shRNAi into mammalian cells.

Article Title: A naturally-monomeric infrared fluorescent protein for protein labeling in vivo
Article Snippet: Paragraph title: Fusion plasmid construction ... To construct the mIFP C-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human β-actin (30), NheI and BglII (cDNA source: Clontech, Mountain View, CA, USA; NM_001101.3); CAF1 (22), AgeI and BspEI (mouse chromatin assembly factor; cDNA source: A. Gunjan, Florida State University, Tallahassee, FL, USA; NM_013733.3); human light chain clathrin (27), NheI and BglII (cDNA source: G. Patterson, National Institutes of Health, Bethesda, MD, USA; NM_001834.2); human endosomes (26), NheI and BspEI (human RhoB GTPase; cDNA source: Clontech; NM_004040.2); human fibrillarin (19), AgeI and BspEI (cDNA source: Evrogen, Moscow, Russia; NM_001436.3); H2B (10), BglII and NheI (human histone 2B, cDNA source: G. Patterson, NIH; NM_021058.3); human lamin A/C (30), NheI and BglII (cDNA source: D. Gilbert, Florida State University; NM_170707.2); human lasp1 (22) NheI and BglII (cDNA source: Origene; NM_006148.3); human myotilin (26), AgeI and BspEI (cDNA source: Origene; NM_006790.2); human Rab4a (19), BglII and BamHI (cDNA source: V. Allen, University of Manchester, Manchester, UK; NM_004578.3); rat sEpsin (30) NheI and BglII (cDNA source: Origene; NM_019585.3); human α-tubulin (30), NheI and BglII (cDNA source: Clontech; NM_006082); human vinculin (35) NheI and EcoRI (cDNA source: Origene; NM_003373.3).

Gel Purification:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: To generate fusion vectors, the appropriate cloning vector and an EGFP fusion vector were digested, either sequentially or doubly, with the appropriate enzymes and ligated together after gel purification. .. To prepare mTFP1 and mWasabi C-terminal fusions, the following digests were performed: human β-actin, NheI and BglII (Clontech); human α-tubulin, NheI and BglII (Clontech); human light chain clathrin, NheI and BglII (George Patterson, NIH); human lamin B1, NheI and BglII (George Patterson, NIH); human fibrillarin, AgeI and BglII (Evrogen); human vinculin, NheI and EcoRI (Open Biosystems, Huntsville, AL); peroximal targeting signal 1 (PTS1 – peroxisomes), AgeI and BspEI (Clontech); chicken protein tyrosine kinase 2, AgeI and BglII (Clare Waterman-Storer, NIH); human annexin (A4), AgeI and BspEI (Alen Piljic, EMBL, Heidelberg); human RhoB GTPase with an N-terminal c-Myc epitope tag (endosomes), AgeI and BspEI (Clontech); and the 20-amino acid farnesylation signal from c-Ha-Ras, AgeI and BspEI (membrane, Clontech).

Article Title: Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy
Article Snippet: To construct the mEos4a/b fusions to the C-terminus of the fluorescent protein (number of linker amino acids in parenthesis), the subsequent digests were performed: human lamin A (18), NheI and BglII (cDNA source: D. Gilbert, FSU; NM_170707.2); human α-tubulin (18), NheI and BglII (cDNA source: Clontech; NM_006082); human H2B (6), BglII and NheI (histones, cDNA source: G. Patterson, NIH; NM_021058.3). .. Following digestion with the appropriate restriction enzymes and gel purification, the plasmids were ligated together with the similarly digested and gel-purified mEos4a or mEos4b cloning vector and purified using a Plasmid Maxiprep kit (Qiagen).

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    TaKaRa recombinant wild type bglii rbgliiwt
    Chromatogram of transglycosylation products by <t>rBGLIIwt.</t> HPLC analysis of transglycosylation products using purified recombinant wild-type <t>BGLII</t> in E. coli . a The chromatogram generated by the Prominence HPLC system. In the standard chromatogram, G4, G3, G2, and G1 represent cellotetraose, cellotriose, cellobiose, and glucose peaks, respectively. Other β-disaccharides, α-sophorose, laminaribiose, and gentiobiose are represented by Sp, Lm, and Ge, respectively. b The chromatogram of size exclusion chromatography. Cello-oligosaccharides were used as the standard substance. Putative transglycosylation products are indicated by arrows . Asterisk refers to the peak of a buffer component in the reactant
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    TaKaRa bglii sali site
    Splicing regulation of MBNL and CELF proteins. ( A ) Structure of chloride channel minigenes. Both human CLCN1/ClC-1 and mouse Clcn1 minigenes were subcloned between the <t>BglII</t> and <t>SalI</t> sites of pEGFP-C1. Black boxes represent exons of the minigenes. Arrows indicate the position of primers used in the splicing assays. Exon 6B is a human-specific exon and is absent in Clcn1 . ( B ) Splicing regulation of Clcn1 by MBNL and CELF proteins. Representative results of cellular splicing assays using the Clcn1 minigene in COS-7 cells. The upper bands correspond to a splice product containing exon 7A, whereas lower bands correspond to a splice product lacking exon 7A. Bar chart shows quantified results of exon 7A inclusion (mean ± SD, n = 3). Statistical significance was analyzed by analysis of variance (ANOVA) and Dunnett's multiple comparisons. All MBNL proteins and CELF proteins except for CUG-BP and ETR-3 showed significant differences (* P
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    Chromatogram of transglycosylation products by rBGLIIwt. HPLC analysis of transglycosylation products using purified recombinant wild-type BGLII in E. coli . a The chromatogram generated by the Prominence HPLC system. In the standard chromatogram, G4, G3, G2, and G1 represent cellotetraose, cellotriose, cellobiose, and glucose peaks, respectively. Other β-disaccharides, α-sophorose, laminaribiose, and gentiobiose are represented by Sp, Lm, and Ge, respectively. b The chromatogram of size exclusion chromatography. Cello-oligosaccharides were used as the standard substance. Putative transglycosylation products are indicated by arrows . Asterisk refers to the peak of a buffer component in the reactant

    Journal: Biotechnology for Biofuels

    Article Title: The impact of a single-nucleotide mutation of bgl2 on cellulase induction in a Trichoderma reesei mutant

    doi: 10.1186/s13068-015-0420-y

    Figure Lengend Snippet: Chromatogram of transglycosylation products by rBGLIIwt. HPLC analysis of transglycosylation products using purified recombinant wild-type BGLII in E. coli . a The chromatogram generated by the Prominence HPLC system. In the standard chromatogram, G4, G3, G2, and G1 represent cellotetraose, cellotriose, cellobiose, and glucose peaks, respectively. Other β-disaccharides, α-sophorose, laminaribiose, and gentiobiose are represented by Sp, Lm, and Ge, respectively. b The chromatogram of size exclusion chromatography. Cello-oligosaccharides were used as the standard substance. Putative transglycosylation products are indicated by arrows . Asterisk refers to the peak of a buffer component in the reactant

    Article Snippet: After cultivation, cell lysate of harvested E. coli cells was prepared using xTracter Buffer (Takara-Bio, Shiga, Japan) and recombinant wild-type BGLII (rBGLIIwt) was purified by TALON® Metal Affinity Resins (Takara-Bio, Shiga, Japan) as described in the instructions.

    Techniques: High Performance Liquid Chromatography, Purification, Recombinant, Generated, Size-exclusion Chromatography

    Schematic diagram of vector construction. 1, PCR products of upstream homologous arm 2A with AscI and BglII, cloned into the pMD18-T vector and named pMD18-T-2A. pMD18-T-2A was digested with AscI and BglII endonucleases and inserted into the pGBHCupp

    Journal: Applied and Environmental Microbiology

    Article Title: Chromosomal Insertions in the Lactobacillus caseiupp Gene That Are Useful for Vaccine Expression

    doi: 10.1128/AEM.00175-14

    Figure Lengend Snippet: Schematic diagram of vector construction. 1, PCR products of upstream homologous arm 2A with AscI and BglII, cloned into the pMD18-T vector and named pMD18-T-2A. pMD18-T-2A was digested with AscI and BglII endonucleases and inserted into the pGBHCupp

    Article Snippet: The pMD18-T-2A plasmid was digested with BglII (TaKaRa) and AscI (NEB).

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Clone Assay

    Verification of pGL3-Enhancer-PPARγ2 (625 bp)-Luc plasmid by electrophoresis. Lane 1: DNA marker DL2000. Lane 2: Inserting fragment from pGL3-PPARγ2 (625 bp)-Luc digested by KpnI and BglII (625 bp). Lane 3: Vector fragment from pGL3-Enhancer-Luc digested by KpnI and BglII (5064 bp). Lane 4: The constructed pGL3-Enhancer-PPARγ2 (625 bp)-Luc plasmid (5689 bp). Lane 5: The constructed pGL3-Enhancer-PPARγ2 (625 bp)-Luc digested by KpnI and BglII (5064/625 bp). Lane 6: DNA marker III.

    Journal: Frontiers in Pharmacology

    Article Title: Lotus Leaf Aqueous Extract Reduces Visceral Fat Mass and Ameliorates Insulin Resistance in HFD-Induced Obese Rats by Regulating PPARγ2 Expression

    doi: 10.3389/fphar.2017.00409

    Figure Lengend Snippet: Verification of pGL3-Enhancer-PPARγ2 (625 bp)-Luc plasmid by electrophoresis. Lane 1: DNA marker DL2000. Lane 2: Inserting fragment from pGL3-PPARγ2 (625 bp)-Luc digested by KpnI and BglII (625 bp). Lane 3: Vector fragment from pGL3-Enhancer-Luc digested by KpnI and BglII (5064 bp). Lane 4: The constructed pGL3-Enhancer-PPARγ2 (625 bp)-Luc plasmid (5689 bp). Lane 5: The constructed pGL3-Enhancer-PPARγ2 (625 bp)-Luc digested by KpnI and BglII (5064/625 bp). Lane 6: DNA marker III.

    Article Snippet: In brief, pGL3-PPARγ2 (625 bp)-Luc, which contained the human PPARγ2 gene 5′-promoter fragment spanning -615 to +10 bp (+1 indicates the transcription start site) and was constructed previously in our laboratory, was digested by the restriction enzymes KpnI and BglII (Takara, Japan) to get the 625 bp PPARγ2 gene promoter inserting fragment.

    Techniques: Plasmid Preparation, Electrophoresis, Marker, Construct

    Splicing regulation of MBNL and CELF proteins. ( A ) Structure of chloride channel minigenes. Both human CLCN1/ClC-1 and mouse Clcn1 minigenes were subcloned between the BglII and SalI sites of pEGFP-C1. Black boxes represent exons of the minigenes. Arrows indicate the position of primers used in the splicing assays. Exon 6B is a human-specific exon and is absent in Clcn1 . ( B ) Splicing regulation of Clcn1 by MBNL and CELF proteins. Representative results of cellular splicing assays using the Clcn1 minigene in COS-7 cells. The upper bands correspond to a splice product containing exon 7A, whereas lower bands correspond to a splice product lacking exon 7A. Bar chart shows quantified results of exon 7A inclusion (mean ± SD, n = 3). Statistical significance was analyzed by analysis of variance (ANOVA) and Dunnett's multiple comparisons. All MBNL proteins and CELF proteins except for CUG-BP and ETR-3 showed significant differences (* P

    Journal: Nucleic Acids Research

    Article Title: MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1

    doi: 10.1093/nar/gkp681

    Figure Lengend Snippet: Splicing regulation of MBNL and CELF proteins. ( A ) Structure of chloride channel minigenes. Both human CLCN1/ClC-1 and mouse Clcn1 minigenes were subcloned between the BglII and SalI sites of pEGFP-C1. Black boxes represent exons of the minigenes. Arrows indicate the position of primers used in the splicing assays. Exon 6B is a human-specific exon and is absent in Clcn1 . ( B ) Splicing regulation of Clcn1 by MBNL and CELF proteins. Representative results of cellular splicing assays using the Clcn1 minigene in COS-7 cells. The upper bands correspond to a splice product containing exon 7A, whereas lower bands correspond to a splice product lacking exon 7A. Bar chart shows quantified results of exon 7A inclusion (mean ± SD, n = 3). Statistical significance was analyzed by analysis of variance (ANOVA) and Dunnett's multiple comparisons. All MBNL proteins and CELF proteins except for CUG-BP and ETR-3 showed significant differences (* P

    Article Snippet: The minigene fragments in pGEM-T Easy were cleaved by BamHI and SalI and then subcloned into the BglII-SalI site of pEGFP-C1 (Clontech).

    Techniques: