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TaKaRa bspei
Bspei, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Clone Assay:

Article Title: Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids ▿
Article Snippet: .. Expressing cell clones were identified by an immunofluorescence (IF) assay for pUL34 expression 20 h after infection with the UL34-null virus vRR1072(TK+). pRR1238 for constitutive expression of wt pUL34 was constructed by digestion of pRR1072Rep with NcoI and BspEI, treatment with Klenow enzyme in the presence of deoxynucleoside triphosphates (dNTPs) to create blunt ends, and ligation of the UL34-containing fragment into EcoRV-digested pcDNA3. pRR1328 for constitutive expression of CL04 pUL34 was constructed by InFusion (Clontech) cloning of a PCR fragment containing the CL04 pUL34 coding sequence amplified from pRR1162 between the HindIII and XhoI sites of pcDNA3. .. PCR primers used for amplification of the CL04 pUL34 coding sequence were 5′-AGGGAGACCCAAGCTCCATGGCGGGACTGGGCAAG-3′ and 5′-TAGATGCATGCTCGATTATAGGCGCGCGCCAGCAC-3′. pRR1330, in which the coding sequences for amino acids 91 to 228 are deleted from the UL34 gene (Fig. , line 3), was constructed by digestion of pRR1238 with SgrAI and HpaI, treatment with Klenow enzyme in the presence of dNTPs to create blunt ends, and religation of the large fragment of the plasmid. pRR1331, in which the same coding sequences are deleted from the CL04 gene (Fig. , line 4), was created in the same way as pRR1330, except that the parent plasmid was pRR1328. pRR1344, in which amino acids 91 to 275 of wt pUL34 expressed from pcDNA3 are deleted and replaced by the emerin transmembrane domain (Fig. , line 5), was constructed by PCR amplification of sequences coding for amino acids 219 to 254 of emerin and ligation of the BspEI- and XbaI-digested PCR product between the SgrAI and XbaI sites of pRR1238.

Article Title: High-mobility group box 1 links sensing of reactive oxygen species by huntingtin to its nuclear entry
Article Snippet: .. Double-stranded synthetic DNA oligonucleotides (Integrated DNA Technologies) encoding the PY-NLS and IVS sequence of huntingtin containing BspEI and Acc65I overhangs were cloned between BspEI/Acc65I sites of peYFPN1 (BD Biosciences/Clontech) to generate huntingtin PY-NLS-eYFPN1 and huntingtin IVS-eYFPN1 plasmids. ..

Amplification:

Article Title: Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids ▿
Article Snippet: .. Expressing cell clones were identified by an immunofluorescence (IF) assay for pUL34 expression 20 h after infection with the UL34-null virus vRR1072(TK+). pRR1238 for constitutive expression of wt pUL34 was constructed by digestion of pRR1072Rep with NcoI and BspEI, treatment with Klenow enzyme in the presence of deoxynucleoside triphosphates (dNTPs) to create blunt ends, and ligation of the UL34-containing fragment into EcoRV-digested pcDNA3. pRR1328 for constitutive expression of CL04 pUL34 was constructed by InFusion (Clontech) cloning of a PCR fragment containing the CL04 pUL34 coding sequence amplified from pRR1162 between the HindIII and XhoI sites of pcDNA3. .. PCR primers used for amplification of the CL04 pUL34 coding sequence were 5′-AGGGAGACCCAAGCTCCATGGCGGGACTGGGCAAG-3′ and 5′-TAGATGCATGCTCGATTATAGGCGCGCGCCAGCAC-3′. pRR1330, in which the coding sequences for amino acids 91 to 228 are deleted from the UL34 gene (Fig. , line 3), was constructed by digestion of pRR1238 with SgrAI and HpaI, treatment with Klenow enzyme in the presence of dNTPs to create blunt ends, and religation of the large fragment of the plasmid. pRR1331, in which the same coding sequences are deleted from the CL04 gene (Fig. , line 4), was created in the same way as pRR1330, except that the parent plasmid was pRR1328. pRR1344, in which amino acids 91 to 275 of wt pUL34 expressed from pcDNA3 are deleted and replaced by the emerin transmembrane domain (Fig. , line 5), was constructed by PCR amplification of sequences coding for amino acids 219 to 254 of emerin and ligation of the BspEI- and XbaI-digested PCR product between the SgrAI and XbaI sites of pRR1238.

Ligation:

Article Title: Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids ▿
Article Snippet: .. Expressing cell clones were identified by an immunofluorescence (IF) assay for pUL34 expression 20 h after infection with the UL34-null virus vRR1072(TK+). pRR1238 for constitutive expression of wt pUL34 was constructed by digestion of pRR1072Rep with NcoI and BspEI, treatment with Klenow enzyme in the presence of deoxynucleoside triphosphates (dNTPs) to create blunt ends, and ligation of the UL34-containing fragment into EcoRV-digested pcDNA3. pRR1328 for constitutive expression of CL04 pUL34 was constructed by InFusion (Clontech) cloning of a PCR fragment containing the CL04 pUL34 coding sequence amplified from pRR1162 between the HindIII and XhoI sites of pcDNA3. .. PCR primers used for amplification of the CL04 pUL34 coding sequence were 5′-AGGGAGACCCAAGCTCCATGGCGGGACTGGGCAAG-3′ and 5′-TAGATGCATGCTCGATTATAGGCGCGCGCCAGCAC-3′. pRR1330, in which the coding sequences for amino acids 91 to 228 are deleted from the UL34 gene (Fig. , line 3), was constructed by digestion of pRR1238 with SgrAI and HpaI, treatment with Klenow enzyme in the presence of dNTPs to create blunt ends, and religation of the large fragment of the plasmid. pRR1331, in which the same coding sequences are deleted from the CL04 gene (Fig. , line 4), was created in the same way as pRR1330, except that the parent plasmid was pRR1328. pRR1344, in which amino acids 91 to 275 of wt pUL34 expressed from pcDNA3 are deleted and replaced by the emerin transmembrane domain (Fig. , line 5), was constructed by PCR amplification of sequences coding for amino acids 219 to 254 of emerin and ligation of the BspEI- and XbaI-digested PCR product between the SgrAI and XbaI sites of pRR1238.

Mutagenesis:

Article Title: Role of the Terminal Domains in Sodium Channel Localization
Article Snippet: .. To generate Nav 1.2-EGFP, the EGFP sequence between NheI and BspEI was cut from the pEGFP-C1 plasmid (Clontech Laboratories, Palo Alto, CA), and the construct was inserted in frame into the 5’ noncoding sequence of the Nav 1.2-FLAG clone behind the promoters and between NheI and BspEI sites created with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). .. Primers used to create the NheI site were 5’-GAGACCGGAAGCTAGCTACCGAGCTCCGGAGG-3 ’ and 5’-CCTCCGGAGCTCGGTAGCTAGCTTCCGGTCTC-3’, and primers used to create the BspEI site were 5 ’-GGTACCGAGCTCCGGAGGTCCACTAGTAAC-3 ’ and 5’-GTTACTAGTGGACCTCCGGAGCTCGGTACC-3’.

Construct:

Article Title: Role of the Terminal Domains in Sodium Channel Localization
Article Snippet: .. To generate Nav 1.2-EGFP, the EGFP sequence between NheI and BspEI was cut from the pEGFP-C1 plasmid (Clontech Laboratories, Palo Alto, CA), and the construct was inserted in frame into the 5’ noncoding sequence of the Nav 1.2-FLAG clone behind the promoters and between NheI and BspEI sites created with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). .. Primers used to create the NheI site were 5’-GAGACCGGAAGCTAGCTACCGAGCTCCGGAGG-3 ’ and 5’-CCTCCGGAGCTCGGTAGCTAGCTTCCGGTCTC-3’, and primers used to create the BspEI site were 5 ’-GGTACCGAGCTCCGGAGGTCCACTAGTAAC-3 ’ and 5’-GTTACTAGTGGACCTCCGGAGCTCGGTACC-3’.

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).

Article Title: mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities
Article Snippet: .. The purified PCR products were then digested and ligated into pEGFP-actin (Clontech), whose FP-coding gene has been previously removed by the same restriction enzymes. pcFP-actinin constructs were generated in a similar way using a 5′ primer encoding an AgeI site, a 3′ primer encoding a BspEI, and the pactinin-EGFP vector (Clontech). .. Plasmid DNA for transfection was prepared using the GeneJET™ Plasmid Miniprep Kit (Fermentas) or using the Plasmid Maxi kit (QIAGEN, Valencia, CA).

Article Title: Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids ▿
Article Snippet: .. Expressing cell clones were identified by an immunofluorescence (IF) assay for pUL34 expression 20 h after infection with the UL34-null virus vRR1072(TK+). pRR1238 for constitutive expression of wt pUL34 was constructed by digestion of pRR1072Rep with NcoI and BspEI, treatment with Klenow enzyme in the presence of deoxynucleoside triphosphates (dNTPs) to create blunt ends, and ligation of the UL34-containing fragment into EcoRV-digested pcDNA3. pRR1328 for constitutive expression of CL04 pUL34 was constructed by InFusion (Clontech) cloning of a PCR fragment containing the CL04 pUL34 coding sequence amplified from pRR1162 between the HindIII and XhoI sites of pcDNA3. .. PCR primers used for amplification of the CL04 pUL34 coding sequence were 5′-AGGGAGACCCAAGCTCCATGGCGGGACTGGGCAAG-3′ and 5′-TAGATGCATGCTCGATTATAGGCGCGCGCCAGCAC-3′. pRR1330, in which the coding sequences for amino acids 91 to 228 are deleted from the UL34 gene (Fig. , line 3), was constructed by digestion of pRR1238 with SgrAI and HpaI, treatment with Klenow enzyme in the presence of dNTPs to create blunt ends, and religation of the large fragment of the plasmid. pRR1331, in which the same coding sequences are deleted from the CL04 gene (Fig. , line 4), was created in the same way as pRR1330, except that the parent plasmid was pRR1328. pRR1344, in which amino acids 91 to 275 of wt pUL34 expressed from pcDNA3 are deleted and replaced by the emerin transmembrane domain (Fig. , line 5), was constructed by PCR amplification of sequences coding for amino acids 219 to 254 of emerin and ligation of the BspEI- and XbaI-digested PCR product between the SgrAI and XbaI sites of pRR1238.

Purification:

Article Title: mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities
Article Snippet: .. The purified PCR products were then digested and ligated into pEGFP-actin (Clontech), whose FP-coding gene has been previously removed by the same restriction enzymes. pcFP-actinin constructs were generated in a similar way using a 5′ primer encoding an AgeI site, a 3′ primer encoding a BspEI, and the pactinin-EGFP vector (Clontech). .. Plasmid DNA for transfection was prepared using the GeneJET™ Plasmid Miniprep Kit (Fermentas) or using the Plasmid Maxi kit (QIAGEN, Valencia, CA).

Sequencing:

Article Title: Role of the Terminal Domains in Sodium Channel Localization
Article Snippet: .. To generate Nav 1.2-EGFP, the EGFP sequence between NheI and BspEI was cut from the pEGFP-C1 plasmid (Clontech Laboratories, Palo Alto, CA), and the construct was inserted in frame into the 5’ noncoding sequence of the Nav 1.2-FLAG clone behind the promoters and between NheI and BspEI sites created with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). .. Primers used to create the NheI site were 5’-GAGACCGGAAGCTAGCTACCGAGCTCCGGAGG-3 ’ and 5’-CCTCCGGAGCTCGGTAGCTAGCTTCCGGTCTC-3’, and primers used to create the BspEI site were 5 ’-GGTACCGAGCTCCGGAGGTCCACTAGTAAC-3 ’ and 5’-GTTACTAGTGGACCTCCGGAGCTCGGTACC-3’.

Article Title: Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids ▿
Article Snippet: .. Expressing cell clones were identified by an immunofluorescence (IF) assay for pUL34 expression 20 h after infection with the UL34-null virus vRR1072(TK+). pRR1238 for constitutive expression of wt pUL34 was constructed by digestion of pRR1072Rep with NcoI and BspEI, treatment with Klenow enzyme in the presence of deoxynucleoside triphosphates (dNTPs) to create blunt ends, and ligation of the UL34-containing fragment into EcoRV-digested pcDNA3. pRR1328 for constitutive expression of CL04 pUL34 was constructed by InFusion (Clontech) cloning of a PCR fragment containing the CL04 pUL34 coding sequence amplified from pRR1162 between the HindIII and XhoI sites of pcDNA3. .. PCR primers used for amplification of the CL04 pUL34 coding sequence were 5′-AGGGAGACCCAAGCTCCATGGCGGGACTGGGCAAG-3′ and 5′-TAGATGCATGCTCGATTATAGGCGCGCGCCAGCAC-3′. pRR1330, in which the coding sequences for amino acids 91 to 228 are deleted from the UL34 gene (Fig. , line 3), was constructed by digestion of pRR1238 with SgrAI and HpaI, treatment with Klenow enzyme in the presence of dNTPs to create blunt ends, and religation of the large fragment of the plasmid. pRR1331, in which the same coding sequences are deleted from the CL04 gene (Fig. , line 4), was created in the same way as pRR1330, except that the parent plasmid was pRR1328. pRR1344, in which amino acids 91 to 275 of wt pUL34 expressed from pcDNA3 are deleted and replaced by the emerin transmembrane domain (Fig. , line 5), was constructed by PCR amplification of sequences coding for amino acids 219 to 254 of emerin and ligation of the BspEI- and XbaI-digested PCR product between the SgrAI and XbaI sites of pRR1238.

Article Title: High-mobility group box 1 links sensing of reactive oxygen species by huntingtin to its nuclear entry
Article Snippet: .. Double-stranded synthetic DNA oligonucleotides (Integrated DNA Technologies) encoding the PY-NLS and IVS sequence of huntingtin containing BspEI and Acc65I overhangs were cloned between BspEI/Acc65I sites of peYFPN1 (BD Biosciences/Clontech) to generate huntingtin PY-NLS-eYFPN1 and huntingtin IVS-eYFPN1 plasmids. ..

Generated:

Article Title: mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities
Article Snippet: .. The purified PCR products were then digested and ligated into pEGFP-actin (Clontech), whose FP-coding gene has been previously removed by the same restriction enzymes. pcFP-actinin constructs were generated in a similar way using a 5′ primer encoding an AgeI site, a 3′ primer encoding a BspEI, and the pactinin-EGFP vector (Clontech). .. Plasmid DNA for transfection was prepared using the GeneJET™ Plasmid Miniprep Kit (Fermentas) or using the Plasmid Maxi kit (QIAGEN, Valencia, CA).

Infection:

Article Title: Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids ▿
Article Snippet: .. Expressing cell clones were identified by an immunofluorescence (IF) assay for pUL34 expression 20 h after infection with the UL34-null virus vRR1072(TK+). pRR1238 for constitutive expression of wt pUL34 was constructed by digestion of pRR1072Rep with NcoI and BspEI, treatment with Klenow enzyme in the presence of deoxynucleoside triphosphates (dNTPs) to create blunt ends, and ligation of the UL34-containing fragment into EcoRV-digested pcDNA3. pRR1328 for constitutive expression of CL04 pUL34 was constructed by InFusion (Clontech) cloning of a PCR fragment containing the CL04 pUL34 coding sequence amplified from pRR1162 between the HindIII and XhoI sites of pcDNA3. .. PCR primers used for amplification of the CL04 pUL34 coding sequence were 5′-AGGGAGACCCAAGCTCCATGGCGGGACTGGGCAAG-3′ and 5′-TAGATGCATGCTCGATTATAGGCGCGCGCCAGCAC-3′. pRR1330, in which the coding sequences for amino acids 91 to 228 are deleted from the UL34 gene (Fig. , line 3), was constructed by digestion of pRR1238 with SgrAI and HpaI, treatment with Klenow enzyme in the presence of dNTPs to create blunt ends, and religation of the large fragment of the plasmid. pRR1331, in which the same coding sequences are deleted from the CL04 gene (Fig. , line 4), was created in the same way as pRR1330, except that the parent plasmid was pRR1328. pRR1344, in which amino acids 91 to 275 of wt pUL34 expressed from pcDNA3 are deleted and replaced by the emerin transmembrane domain (Fig. , line 5), was constructed by PCR amplification of sequences coding for amino acids 219 to 254 of emerin and ligation of the BspEI- and XbaI-digested PCR product between the SgrAI and XbaI sites of pRR1238.

Expressing:

Article Title: Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids ▿
Article Snippet: .. Expressing cell clones were identified by an immunofluorescence (IF) assay for pUL34 expression 20 h after infection with the UL34-null virus vRR1072(TK+). pRR1238 for constitutive expression of wt pUL34 was constructed by digestion of pRR1072Rep with NcoI and BspEI, treatment with Klenow enzyme in the presence of deoxynucleoside triphosphates (dNTPs) to create blunt ends, and ligation of the UL34-containing fragment into EcoRV-digested pcDNA3. pRR1328 for constitutive expression of CL04 pUL34 was constructed by InFusion (Clontech) cloning of a PCR fragment containing the CL04 pUL34 coding sequence amplified from pRR1162 between the HindIII and XhoI sites of pcDNA3. .. PCR primers used for amplification of the CL04 pUL34 coding sequence were 5′-AGGGAGACCCAAGCTCCATGGCGGGACTGGGCAAG-3′ and 5′-TAGATGCATGCTCGATTATAGGCGCGCGCCAGCAC-3′. pRR1330, in which the coding sequences for amino acids 91 to 228 are deleted from the UL34 gene (Fig. , line 3), was constructed by digestion of pRR1238 with SgrAI and HpaI, treatment with Klenow enzyme in the presence of dNTPs to create blunt ends, and religation of the large fragment of the plasmid. pRR1331, in which the same coding sequences are deleted from the CL04 gene (Fig. , line 4), was created in the same way as pRR1330, except that the parent plasmid was pRR1328. pRR1344, in which amino acids 91 to 275 of wt pUL34 expressed from pcDNA3 are deleted and replaced by the emerin transmembrane domain (Fig. , line 5), was constructed by PCR amplification of sequences coding for amino acids 219 to 254 of emerin and ligation of the BspEI- and XbaI-digested PCR product between the SgrAI and XbaI sites of pRR1238.

Polymerase Chain Reaction:

Article Title: Beyond the heterodimer model for mineralocorticoid and glucocorticoid receptor interactions in nuclei and at DNA
Article Snippet: .. The PCR product was inserted into the BspEI and SacII sites of pEGFP-C1 or pmCherry-C1 (Clontech, Mountain View, CA) to obtain pEGFP-C1-rMR or pmCherry-C1-rMR. ..

Article Title: mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities
Article Snippet: .. The purified PCR products were then digested and ligated into pEGFP-actin (Clontech), whose FP-coding gene has been previously removed by the same restriction enzymes. pcFP-actinin constructs were generated in a similar way using a 5′ primer encoding an AgeI site, a 3′ primer encoding a BspEI, and the pactinin-EGFP vector (Clontech). .. Plasmid DNA for transfection was prepared using the GeneJET™ Plasmid Miniprep Kit (Fermentas) or using the Plasmid Maxi kit (QIAGEN, Valencia, CA).

Article Title: Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids ▿
Article Snippet: .. Expressing cell clones were identified by an immunofluorescence (IF) assay for pUL34 expression 20 h after infection with the UL34-null virus vRR1072(TK+). pRR1238 for constitutive expression of wt pUL34 was constructed by digestion of pRR1072Rep with NcoI and BspEI, treatment with Klenow enzyme in the presence of deoxynucleoside triphosphates (dNTPs) to create blunt ends, and ligation of the UL34-containing fragment into EcoRV-digested pcDNA3. pRR1328 for constitutive expression of CL04 pUL34 was constructed by InFusion (Clontech) cloning of a PCR fragment containing the CL04 pUL34 coding sequence amplified from pRR1162 between the HindIII and XhoI sites of pcDNA3. .. PCR primers used for amplification of the CL04 pUL34 coding sequence were 5′-AGGGAGACCCAAGCTCCATGGCGGGACTGGGCAAG-3′ and 5′-TAGATGCATGCTCGATTATAGGCGCGCGCCAGCAC-3′. pRR1330, in which the coding sequences for amino acids 91 to 228 are deleted from the UL34 gene (Fig. , line 3), was constructed by digestion of pRR1238 with SgrAI and HpaI, treatment with Klenow enzyme in the presence of dNTPs to create blunt ends, and religation of the large fragment of the plasmid. pRR1331, in which the same coding sequences are deleted from the CL04 gene (Fig. , line 4), was created in the same way as pRR1330, except that the parent plasmid was pRR1328. pRR1344, in which amino acids 91 to 275 of wt pUL34 expressed from pcDNA3 are deleted and replaced by the emerin transmembrane domain (Fig. , line 5), was constructed by PCR amplification of sequences coding for amino acids 219 to 254 of emerin and ligation of the BspEI- and XbaI-digested PCR product between the SgrAI and XbaI sites of pRR1238.

Immunofluorescence:

Article Title: Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids ▿
Article Snippet: .. Expressing cell clones were identified by an immunofluorescence (IF) assay for pUL34 expression 20 h after infection with the UL34-null virus vRR1072(TK+). pRR1238 for constitutive expression of wt pUL34 was constructed by digestion of pRR1072Rep with NcoI and BspEI, treatment with Klenow enzyme in the presence of deoxynucleoside triphosphates (dNTPs) to create blunt ends, and ligation of the UL34-containing fragment into EcoRV-digested pcDNA3. pRR1328 for constitutive expression of CL04 pUL34 was constructed by InFusion (Clontech) cloning of a PCR fragment containing the CL04 pUL34 coding sequence amplified from pRR1162 between the HindIII and XhoI sites of pcDNA3. .. PCR primers used for amplification of the CL04 pUL34 coding sequence were 5′-AGGGAGACCCAAGCTCCATGGCGGGACTGGGCAAG-3′ and 5′-TAGATGCATGCTCGATTATAGGCGCGCGCCAGCAC-3′. pRR1330, in which the coding sequences for amino acids 91 to 228 are deleted from the UL34 gene (Fig. , line 3), was constructed by digestion of pRR1238 with SgrAI and HpaI, treatment with Klenow enzyme in the presence of dNTPs to create blunt ends, and religation of the large fragment of the plasmid. pRR1331, in which the same coding sequences are deleted from the CL04 gene (Fig. , line 4), was created in the same way as pRR1330, except that the parent plasmid was pRR1328. pRR1344, in which amino acids 91 to 275 of wt pUL34 expressed from pcDNA3 are deleted and replaced by the emerin transmembrane domain (Fig. , line 5), was constructed by PCR amplification of sequences coding for amino acids 219 to 254 of emerin and ligation of the BspEI- and XbaI-digested PCR product between the SgrAI and XbaI sites of pRR1238.

Plasmid Preparation:

Article Title: Role of the Terminal Domains in Sodium Channel Localization
Article Snippet: .. To generate Nav 1.2-EGFP, the EGFP sequence between NheI and BspEI was cut from the pEGFP-C1 plasmid (Clontech Laboratories, Palo Alto, CA), and the construct was inserted in frame into the 5’ noncoding sequence of the Nav 1.2-FLAG clone behind the promoters and between NheI and BspEI sites created with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). .. Primers used to create the NheI site were 5’-GAGACCGGAAGCTAGCTACCGAGCTCCGGAGG-3 ’ and 5’-CCTCCGGAGCTCGGTAGCTAGCTTCCGGTCTC-3’, and primers used to create the BspEI site were 5 ’-GGTACCGAGCTCCGGAGGTCCACTAGTAAC-3 ’ and 5’-GTTACTAGTGGACCTCCGGAGCTCGGTACC-3’.

Article Title: mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities
Article Snippet: .. The purified PCR products were then digested and ligated into pEGFP-actin (Clontech), whose FP-coding gene has been previously removed by the same restriction enzymes. pcFP-actinin constructs were generated in a similar way using a 5′ primer encoding an AgeI site, a 3′ primer encoding a BspEI, and the pactinin-EGFP vector (Clontech). .. Plasmid DNA for transfection was prepared using the GeneJET™ Plasmid Miniprep Kit (Fermentas) or using the Plasmid Maxi kit (QIAGEN, Valencia, CA).

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    TaKaRa bspei
    Bspei, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bspei/product/TaKaRa
    Average 91 stars, based on 19 article reviews
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    TaKaRa bspei acc65i sites
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