Structured Review

OriGene bamhi
Bamhi, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Clone Assay:

Article Title: A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum
Article Snippet: To obtain targeting fusion vectors, the appropriate cloning vector and a previously assembled EGFP or mEmerald fusion vector were digested, either sequentially or doubly, with the appropriate enzymes and ligated together after gel purification. .. To prepare mNeonGreen N-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (cDNA source, Tom Keller, Florida State University (FSU), Tallahassee, FL, U.S.A.; NM_001130005.1); human calnexin, AgeI and NotI (Origene; NM_001746.3); c-src (7), BamHI and EcoRI (chicken c-src cDNA source: Marilyn Resh, Sloan-Kettering, New York; XM_001232484.1); connexin-43 (7), BamHI and NotI (rat C×43 cDNA source: Matthias Falk, Lehigh U; NM_001004099.1); EB3 (7), BglII and BamHI (EB3 cDNA source: Lynne Cassimeris, Lehigh University; NM_012326.2); human keratin 18, EcoRI and NotI (Open Biosystems; NM_199187.1); lamin B1 (10), EcoRI and BamHI (human lamin B1 cDNA source: George Patterson, NIH; NM_005573.2); Lifeact (7), BamHI and NotI (Lifeact cDNA source: IDT); mouse mannosidase 2 (112 N-terminal amino acids, MANNII; 10), NheI and BamHI (cDNA source: Jennifer Lippincott-Schwartz, NIH; NM_008549.2); myosin IIA (14) NheI and BglII (mouse myosin IIA cDNA source: Origene; NM_022410.2); human nucleoporin 50kDa, BamHI and NotI (NUP50 cDNA source: Origene; NM_007172.2); human pyruvate dehydrogenase, AgeI and NotI (human PDHA1 cDNA source: Origene; NM_000284); human peroxisomal membrane protein, NotI and AgeI (PMP cDNA source: Origene; NM_018663.1); human MAP Tau (10), AgeI and NotI (MAP Tau cDNA source: Origene; NM_016841); human TfR (20), BamHI and NotI (transferrin receptor cDNA source: George Patterson, NIH; NM_NM_003234); human TPX2 (10), AgeI and NotI (TPX2 cDNA source: Patricia Wadsworth, University of Massachusetts, Amherst; NM_012112.4); mouse VASP (10), NheI and BamHI (cDNA source: Clare Waterman, NIH; NM_009499); vascular epithelial cadherin (10), AgeI and NotI (human VE cadherin cDNA source: Origene; NM_001795.3), vimentin (7), BamHI and NotI (human vimentin cDNA source: Robert Goldman, Northwestern University; NM_003380.3), zyxin (6), BamHI and NotI (human zyxin cDNA source: Origene; NM_003461).

Article Title: Pathogenic variants in the AFG3L2 proteolytic domain cause SCA28 through haploinsufficiency and proteostatic stress-driven OMA1 activation
Article Snippet: .. Complementary oligonucleotides (AFG3L2 FWD 5′ gatcgCTCCTCGTGCCTGGCGGCGTg 3′ and AFG3L2 REV 5′ aaaacACGCCGCCAGGCACGAGGAGc 3′) for gRNA were annealed and cloned into BamHI and BsmBI sites of pCas-Guide-EF1a-GFP CRISPR/Cas9 All-in-One vector (GE10018, OriGene). .. HEK 293 T cells were transfected with the pCas-Guide-EF1a-GFP vector containing the AFG3L2 targeting sequence, and 48 hours after transfection, Green Fluorescent Protein (GFP)-positive cells were sorted through fluorescence-activated cell sorting.

Transfection:

Article Title: A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum
Article Snippet: To prepare mNeonGreen N-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (cDNA source, Tom Keller, Florida State University (FSU), Tallahassee, FL, U.S.A.; NM_001130005.1); human calnexin, AgeI and NotI (Origene; NM_001746.3); c-src (7), BamHI and EcoRI (chicken c-src cDNA source: Marilyn Resh, Sloan-Kettering, New York; XM_001232484.1); connexin-43 (7), BamHI and NotI (rat C×43 cDNA source: Matthias Falk, Lehigh U; NM_001004099.1); EB3 (7), BglII and BamHI (EB3 cDNA source: Lynne Cassimeris, Lehigh University; NM_012326.2); human keratin 18, EcoRI and NotI (Open Biosystems; NM_199187.1); lamin B1 (10), EcoRI and BamHI (human lamin B1 cDNA source: George Patterson, NIH; NM_005573.2); Lifeact (7), BamHI and NotI (Lifeact cDNA source: IDT); mouse mannosidase 2 (112 N-terminal amino acids, MANNII; 10), NheI and BamHI (cDNA source: Jennifer Lippincott-Schwartz, NIH; NM_008549.2); myosin IIA (14) NheI and BglII (mouse myosin IIA cDNA source: Origene; NM_022410.2); human nucleoporin 50kDa, BamHI and NotI (NUP50 cDNA source: Origene; NM_007172.2); human pyruvate dehydrogenase, AgeI and NotI (human PDHA1 cDNA source: Origene; NM_000284); human peroxisomal membrane protein, NotI and AgeI (PMP cDNA source: Origene; NM_018663.1); human MAP Tau (10), AgeI and NotI (MAP Tau cDNA source: Origene; NM_016841); human TfR (20), BamHI and NotI (transferrin receptor cDNA source: George Patterson, NIH; NM_NM_003234); human TPX2 (10), AgeI and NotI (TPX2 cDNA source: Patricia Wadsworth, University of Massachusetts, Amherst; NM_012112.4); mouse VASP (10), NheI and BamHI (cDNA source: Clare Waterman, NIH; NM_009499); vascular epithelial cadherin (10), AgeI and NotI (human VE cadherin cDNA source: Origene; NM_001795.3), vimentin (7), BamHI and NotI (human vimentin cDNA source: Robert Goldman, Northwestern University; NM_003380.3), zyxin (6), BamHI and NotI (human zyxin cDNA source: Origene; NM_003461). .. All DNA for transfection was prepared using the Plasmid Maxi kit (QIAGEN).

Article Title: Pathogenic variants in the AFG3L2 proteolytic domain cause SCA28 through haploinsufficiency and proteostatic stress-driven OMA1 activation
Article Snippet: Complementary oligonucleotides (AFG3L2 FWD 5′ gatcgCTCCTCGTGCCTGGCGGCGTg 3′ and AFG3L2 REV 5′ aaaacACGCCGCCAGGCACGAGGAGc 3′) for gRNA were annealed and cloned into BamHI and BsmBI sites of pCas-Guide-EF1a-GFP CRISPR/Cas9 All-in-One vector (GE10018, OriGene). .. HEK 293 T cells were transfected with the pCas-Guide-EF1a-GFP vector containing the AFG3L2 targeting sequence, and 48 hours after transfection, Green Fluorescent Protein (GFP)-positive cells were sorted through fluorescence-activated cell sorting.

shRNA:

Article Title: Myosin 1b Regulates Amino Acid Transport by Associating Transporters with the Apical Plasma Membrane of Kidney Cells
Article Snippet: For collectrin expression, the GFP cassette of pLenti-CMV-GFP-Hygro (Addgene plasmid 17446; [ ]) was excised with BamHI and SalI and replaced with the cDNA of human collectrin (Tmem27 ) prepared by PCR from Tmem27-pCMV6-XL5 (SC122895; Origene, Rockville, MD) with the appropriate restriction sites. .. Lentiviral vectors for the expression of Myo1b-specific shRNA were prepared using the lentiviral vector pLKO.1 puro (Addgene plasmid 8453; [ ]) linearized with the restriction enzymes EcoRI and AgeI and ligated with the following sets of oligonucleotides: #243 (CCG GAG CCA TTC TCT AAT AAA GGC TCT CGA GAG CCT TTA TTA GAG AAT GGC TTT TTT G; AAT TCA AAA AAG CCA TTC TCT AAT AAA GGC TCT CGA GAG CCT TTA TTA GAG AAT GGC T); #628 (CCG GAA GAA TAT TCC TTT GGT AGA TCT CGA GAT CTA CCA AAG GAA TAT TCT TTT TTT G; AATTCAAAAAAAGAA TAT TCC TTT GGT AGA TCT CGA GAT CTA CCA AAG GAA TAT TCT T); and #891 (CCG GAG GAT GGA AGG CTC GAA AGA TCT CGA GAT CTT TCG AGC CTT CCA TCC TTT TTT G; AAT TCA AAA AAG GAT GGA AGG CTC GAA AGA TCT CGA GAT CTT TCG AGC CTT CCA TCC T).

Fluorescence:

Article Title: Pathogenic variants in the AFG3L2 proteolytic domain cause SCA28 through haploinsufficiency and proteostatic stress-driven OMA1 activation
Article Snippet: Complementary oligonucleotides (AFG3L2 FWD 5′ gatcgCTCCTCGTGCCTGGCGGCGTg 3′ and AFG3L2 REV 5′ aaaacACGCCGCCAGGCACGAGGAGc 3′) for gRNA were annealed and cloned into BamHI and BsmBI sites of pCas-Guide-EF1a-GFP CRISPR/Cas9 All-in-One vector (GE10018, OriGene). .. HEK 293 T cells were transfected with the pCas-Guide-EF1a-GFP vector containing the AFG3L2 targeting sequence, and 48 hours after transfection, Green Fluorescent Protein (GFP)-positive cells were sorted through fluorescence-activated cell sorting.

Gel Purification:

Article Title: A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum
Article Snippet: To obtain targeting fusion vectors, the appropriate cloning vector and a previously assembled EGFP or mEmerald fusion vector were digested, either sequentially or doubly, with the appropriate enzymes and ligated together after gel purification. .. To prepare mNeonGreen N-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (cDNA source, Tom Keller, Florida State University (FSU), Tallahassee, FL, U.S.A.; NM_001130005.1); human calnexin, AgeI and NotI (Origene; NM_001746.3); c-src (7), BamHI and EcoRI (chicken c-src cDNA source: Marilyn Resh, Sloan-Kettering, New York; XM_001232484.1); connexin-43 (7), BamHI and NotI (rat C×43 cDNA source: Matthias Falk, Lehigh U; NM_001004099.1); EB3 (7), BglII and BamHI (EB3 cDNA source: Lynne Cassimeris, Lehigh University; NM_012326.2); human keratin 18, EcoRI and NotI (Open Biosystems; NM_199187.1); lamin B1 (10), EcoRI and BamHI (human lamin B1 cDNA source: George Patterson, NIH; NM_005573.2); Lifeact (7), BamHI and NotI (Lifeact cDNA source: IDT); mouse mannosidase 2 (112 N-terminal amino acids, MANNII; 10), NheI and BamHI (cDNA source: Jennifer Lippincott-Schwartz, NIH; NM_008549.2); myosin IIA (14) NheI and BglII (mouse myosin IIA cDNA source: Origene; NM_022410.2); human nucleoporin 50kDa, BamHI and NotI (NUP50 cDNA source: Origene; NM_007172.2); human pyruvate dehydrogenase, AgeI and NotI (human PDHA1 cDNA source: Origene; NM_000284); human peroxisomal membrane protein, NotI and AgeI (PMP cDNA source: Origene; NM_018663.1); human MAP Tau (10), AgeI and NotI (MAP Tau cDNA source: Origene; NM_016841); human TfR (20), BamHI and NotI (transferrin receptor cDNA source: George Patterson, NIH; NM_NM_003234); human TPX2 (10), AgeI and NotI (TPX2 cDNA source: Patricia Wadsworth, University of Massachusetts, Amherst; NM_012112.4); mouse VASP (10), NheI and BamHI (cDNA source: Clare Waterman, NIH; NM_009499); vascular epithelial cadherin (10), AgeI and NotI (human VE cadherin cDNA source: Origene; NM_001795.3), vimentin (7), BamHI and NotI (human vimentin cDNA source: Robert Goldman, Northwestern University; NM_003380.3), zyxin (6), BamHI and NotI (human zyxin cDNA source: Origene; NM_003461).

Construct:

Article Title: A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum
Article Snippet: Fusion Plasmid Construction mNeonGreen fluorescent protein expression vectors were constructed using C1 and N1 (Clontech-style) cloning vectors. .. To prepare mNeonGreen N-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (cDNA source, Tom Keller, Florida State University (FSU), Tallahassee, FL, U.S.A.; NM_001130005.1); human calnexin, AgeI and NotI (Origene; NM_001746.3); c-src (7), BamHI and EcoRI (chicken c-src cDNA source: Marilyn Resh, Sloan-Kettering, New York; XM_001232484.1); connexin-43 (7), BamHI and NotI (rat C×43 cDNA source: Matthias Falk, Lehigh U; NM_001004099.1); EB3 (7), BglII and BamHI (EB3 cDNA source: Lynne Cassimeris, Lehigh University; NM_012326.2); human keratin 18, EcoRI and NotI (Open Biosystems; NM_199187.1); lamin B1 (10), EcoRI and BamHI (human lamin B1 cDNA source: George Patterson, NIH; NM_005573.2); Lifeact (7), BamHI and NotI (Lifeact cDNA source: IDT); mouse mannosidase 2 (112 N-terminal amino acids, MANNII; 10), NheI and BamHI (cDNA source: Jennifer Lippincott-Schwartz, NIH; NM_008549.2); myosin IIA (14) NheI and BglII (mouse myosin IIA cDNA source: Origene; NM_022410.2); human nucleoporin 50kDa, BamHI and NotI (NUP50 cDNA source: Origene; NM_007172.2); human pyruvate dehydrogenase, AgeI and NotI (human PDHA1 cDNA source: Origene; NM_000284); human peroxisomal membrane protein, NotI and AgeI (PMP cDNA source: Origene; NM_018663.1); human MAP Tau (10), AgeI and NotI (MAP Tau cDNA source: Origene; NM_016841); human TfR (20), BamHI and NotI (transferrin receptor cDNA source: George Patterson, NIH; NM_NM_003234); human TPX2 (10), AgeI and NotI (TPX2 cDNA source: Patricia Wadsworth, University of Massachusetts, Amherst; NM_012112.4); mouse VASP (10), NheI and BamHI (cDNA source: Clare Waterman, NIH; NM_009499); vascular epithelial cadherin (10), AgeI and NotI (human VE cadherin cDNA source: Origene; NM_001795.3), vimentin (7), BamHI and NotI (human vimentin cDNA source: Robert Goldman, Northwestern University; NM_003380.3), zyxin (6), BamHI and NotI (human zyxin cDNA source: Origene; NM_003461).

Article Title: Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction
Article Snippet: .. In brief, to construct plasmid pACLTR2 EF1α-hNRTN, the plasmid pACLTR2 EF1α were digested with BamHI filled in with T4 DNA polymerase and then digested with EcoRI to ligate ∼0.6 kb of hNRTN; pCMV6-XL5-hNRTN (Origene) was digested with Xba I and filled in with T4 DNA polymerase and digested with EcoRI to obtain the hNRTN cDNA. .. The recombinant vector AdLTR2 EF1α-hNRTN was produced by homologous recombination of pACLTR2EF1a-hNRTN with pJM17 in C7 cells.

Purification:

Article Title: A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum
Article Snippet: Purified and digested PCR products were ligated into similarly digested EGFP-C1 and EGFP-N1 cloning vector backbones. .. To prepare mNeonGreen N-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (cDNA source, Tom Keller, Florida State University (FSU), Tallahassee, FL, U.S.A.; NM_001130005.1); human calnexin, AgeI and NotI (Origene; NM_001746.3); c-src (7), BamHI and EcoRI (chicken c-src cDNA source: Marilyn Resh, Sloan-Kettering, New York; XM_001232484.1); connexin-43 (7), BamHI and NotI (rat C×43 cDNA source: Matthias Falk, Lehigh U; NM_001004099.1); EB3 (7), BglII and BamHI (EB3 cDNA source: Lynne Cassimeris, Lehigh University; NM_012326.2); human keratin 18, EcoRI and NotI (Open Biosystems; NM_199187.1); lamin B1 (10), EcoRI and BamHI (human lamin B1 cDNA source: George Patterson, NIH; NM_005573.2); Lifeact (7), BamHI and NotI (Lifeact cDNA source: IDT); mouse mannosidase 2 (112 N-terminal amino acids, MANNII; 10), NheI and BamHI (cDNA source: Jennifer Lippincott-Schwartz, NIH; NM_008549.2); myosin IIA (14) NheI and BglII (mouse myosin IIA cDNA source: Origene; NM_022410.2); human nucleoporin 50kDa, BamHI and NotI (NUP50 cDNA source: Origene; NM_007172.2); human pyruvate dehydrogenase, AgeI and NotI (human PDHA1 cDNA source: Origene; NM_000284); human peroxisomal membrane protein, NotI and AgeI (PMP cDNA source: Origene; NM_018663.1); human MAP Tau (10), AgeI and NotI (MAP Tau cDNA source: Origene; NM_016841); human TfR (20), BamHI and NotI (transferrin receptor cDNA source: George Patterson, NIH; NM_NM_003234); human TPX2 (10), AgeI and NotI (TPX2 cDNA source: Patricia Wadsworth, University of Massachusetts, Amherst; NM_012112.4); mouse VASP (10), NheI and BamHI (cDNA source: Clare Waterman, NIH; NM_009499); vascular epithelial cadherin (10), AgeI and NotI (human VE cadherin cDNA source: Origene; NM_001795.3), vimentin (7), BamHI and NotI (human vimentin cDNA source: Robert Goldman, Northwestern University; NM_003380.3), zyxin (6), BamHI and NotI (human zyxin cDNA source: Origene; NM_003461).

Article Title: Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction
Article Snippet: In brief, to construct plasmid pACLTR2 EF1α-hNRTN, the plasmid pACLTR2 EF1α were digested with BamHI filled in with T4 DNA polymerase and then digested with EcoRI to ligate ∼0.6 kb of hNRTN; pCMV6-XL5-hNRTN (Origene) was digested with Xba I and filled in with T4 DNA polymerase and digested with EcoRI to obtain the hNRTN cDNA. .. The titers (particles/mL) of purified vectors were determined by qPCR using primers from the E2 region.

Produced:

Article Title: Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction
Article Snippet: In brief, to construct plasmid pACLTR2 EF1α-hNRTN, the plasmid pACLTR2 EF1α were digested with BamHI filled in with T4 DNA polymerase and then digested with EcoRI to ligate ∼0.6 kb of hNRTN; pCMV6-XL5-hNRTN (Origene) was digested with Xba I and filled in with T4 DNA polymerase and digested with EcoRI to obtain the hNRTN cDNA. .. The recombinant vector AdLTR2 EF1α-hNRTN was produced by homologous recombination of pACLTR2EF1a-hNRTN with pJM17 in C7 cells.

Polymerase Chain Reaction:

Article Title: Myosin 1b Regulates Amino Acid Transport by Associating Transporters with the Apical Plasma Membrane of Kidney Cells
Article Snippet: .. For collectrin expression, the GFP cassette of pLenti-CMV-GFP-Hygro (Addgene plasmid 17446; [ ]) was excised with BamHI and SalI and replaced with the cDNA of human collectrin (Tmem27 ) prepared by PCR from Tmem27-pCMV6-XL5 (SC122895; Origene, Rockville, MD) with the appropriate restriction sites. ..

Article Title: A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum
Article Snippet: Purified and digested PCR products were ligated into similarly digested EGFP-C1 and EGFP-N1 cloning vector backbones. .. To prepare mNeonGreen N-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (cDNA source, Tom Keller, Florida State University (FSU), Tallahassee, FL, U.S.A.; NM_001130005.1); human calnexin, AgeI and NotI (Origene; NM_001746.3); c-src (7), BamHI and EcoRI (chicken c-src cDNA source: Marilyn Resh, Sloan-Kettering, New York; XM_001232484.1); connexin-43 (7), BamHI and NotI (rat C×43 cDNA source: Matthias Falk, Lehigh U; NM_001004099.1); EB3 (7), BglII and BamHI (EB3 cDNA source: Lynne Cassimeris, Lehigh University; NM_012326.2); human keratin 18, EcoRI and NotI (Open Biosystems; NM_199187.1); lamin B1 (10), EcoRI and BamHI (human lamin B1 cDNA source: George Patterson, NIH; NM_005573.2); Lifeact (7), BamHI and NotI (Lifeact cDNA source: IDT); mouse mannosidase 2 (112 N-terminal amino acids, MANNII; 10), NheI and BamHI (cDNA source: Jennifer Lippincott-Schwartz, NIH; NM_008549.2); myosin IIA (14) NheI and BglII (mouse myosin IIA cDNA source: Origene; NM_022410.2); human nucleoporin 50kDa, BamHI and NotI (NUP50 cDNA source: Origene; NM_007172.2); human pyruvate dehydrogenase, AgeI and NotI (human PDHA1 cDNA source: Origene; NM_000284); human peroxisomal membrane protein, NotI and AgeI (PMP cDNA source: Origene; NM_018663.1); human MAP Tau (10), AgeI and NotI (MAP Tau cDNA source: Origene; NM_016841); human TfR (20), BamHI and NotI (transferrin receptor cDNA source: George Patterson, NIH; NM_NM_003234); human TPX2 (10), AgeI and NotI (TPX2 cDNA source: Patricia Wadsworth, University of Massachusetts, Amherst; NM_012112.4); mouse VASP (10), NheI and BamHI (cDNA source: Clare Waterman, NIH; NM_009499); vascular epithelial cadherin (10), AgeI and NotI (human VE cadherin cDNA source: Origene; NM_001795.3), vimentin (7), BamHI and NotI (human vimentin cDNA source: Robert Goldman, Northwestern University; NM_003380.3), zyxin (6), BamHI and NotI (human zyxin cDNA source: Origene; NM_003461).

Amplification:

Article Title: A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum
Article Snippet: The mNeonGreen cDNA was amplified with a 5′ primer encoding an AgeI site and a 3′ primer encoding either a BspEI (C1) or NotI (N1) site for generating cloning vectors to create C-terminal and N-terminal fusions (with regards to the FP), respectively. .. To prepare mNeonGreen N-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (cDNA source, Tom Keller, Florida State University (FSU), Tallahassee, FL, U.S.A.; NM_001130005.1); human calnexin, AgeI and NotI (Origene; NM_001746.3); c-src (7), BamHI and EcoRI (chicken c-src cDNA source: Marilyn Resh, Sloan-Kettering, New York; XM_001232484.1); connexin-43 (7), BamHI and NotI (rat C×43 cDNA source: Matthias Falk, Lehigh U; NM_001004099.1); EB3 (7), BglII and BamHI (EB3 cDNA source: Lynne Cassimeris, Lehigh University; NM_012326.2); human keratin 18, EcoRI and NotI (Open Biosystems; NM_199187.1); lamin B1 (10), EcoRI and BamHI (human lamin B1 cDNA source: George Patterson, NIH; NM_005573.2); Lifeact (7), BamHI and NotI (Lifeact cDNA source: IDT); mouse mannosidase 2 (112 N-terminal amino acids, MANNII; 10), NheI and BamHI (cDNA source: Jennifer Lippincott-Schwartz, NIH; NM_008549.2); myosin IIA (14) NheI and BglII (mouse myosin IIA cDNA source: Origene; NM_022410.2); human nucleoporin 50kDa, BamHI and NotI (NUP50 cDNA source: Origene; NM_007172.2); human pyruvate dehydrogenase, AgeI and NotI (human PDHA1 cDNA source: Origene; NM_000284); human peroxisomal membrane protein, NotI and AgeI (PMP cDNA source: Origene; NM_018663.1); human MAP Tau (10), AgeI and NotI (MAP Tau cDNA source: Origene; NM_016841); human TfR (20), BamHI and NotI (transferrin receptor cDNA source: George Patterson, NIH; NM_NM_003234); human TPX2 (10), AgeI and NotI (TPX2 cDNA source: Patricia Wadsworth, University of Massachusetts, Amherst; NM_012112.4); mouse VASP (10), NheI and BamHI (cDNA source: Clare Waterman, NIH; NM_009499); vascular epithelial cadherin (10), AgeI and NotI (human VE cadherin cDNA source: Origene; NM_001795.3), vimentin (7), BamHI and NotI (human vimentin cDNA source: Robert Goldman, Northwestern University; NM_003380.3), zyxin (6), BamHI and NotI (human zyxin cDNA source: Origene; NM_003461).

Plasmid Preparation:

Article Title: Myosin 1b Regulates Amino Acid Transport by Associating Transporters with the Apical Plasma Membrane of Kidney Cells
Article Snippet: .. For collectrin expression, the GFP cassette of pLenti-CMV-GFP-Hygro (Addgene plasmid 17446; [ ]) was excised with BamHI and SalI and replaced with the cDNA of human collectrin (Tmem27 ) prepared by PCR from Tmem27-pCMV6-XL5 (SC122895; Origene, Rockville, MD) with the appropriate restriction sites. ..

Article Title: A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum
Article Snippet: Paragraph title: Fusion Plasmid Construction ... To prepare mNeonGreen N-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (cDNA source, Tom Keller, Florida State University (FSU), Tallahassee, FL, U.S.A.; NM_001130005.1); human calnexin, AgeI and NotI (Origene; NM_001746.3); c-src (7), BamHI and EcoRI (chicken c-src cDNA source: Marilyn Resh, Sloan-Kettering, New York; XM_001232484.1); connexin-43 (7), BamHI and NotI (rat C×43 cDNA source: Matthias Falk, Lehigh U; NM_001004099.1); EB3 (7), BglII and BamHI (EB3 cDNA source: Lynne Cassimeris, Lehigh University; NM_012326.2); human keratin 18, EcoRI and NotI (Open Biosystems; NM_199187.1); lamin B1 (10), EcoRI and BamHI (human lamin B1 cDNA source: George Patterson, NIH; NM_005573.2); Lifeact (7), BamHI and NotI (Lifeact cDNA source: IDT); mouse mannosidase 2 (112 N-terminal amino acids, MANNII; 10), NheI and BamHI (cDNA source: Jennifer Lippincott-Schwartz, NIH; NM_008549.2); myosin IIA (14) NheI and BglII (mouse myosin IIA cDNA source: Origene; NM_022410.2); human nucleoporin 50kDa, BamHI and NotI (NUP50 cDNA source: Origene; NM_007172.2); human pyruvate dehydrogenase, AgeI and NotI (human PDHA1 cDNA source: Origene; NM_000284); human peroxisomal membrane protein, NotI and AgeI (PMP cDNA source: Origene; NM_018663.1); human MAP Tau (10), AgeI and NotI (MAP Tau cDNA source: Origene; NM_016841); human TfR (20), BamHI and NotI (transferrin receptor cDNA source: George Patterson, NIH; NM_NM_003234); human TPX2 (10), AgeI and NotI (TPX2 cDNA source: Patricia Wadsworth, University of Massachusetts, Amherst; NM_012112.4); mouse VASP (10), NheI and BamHI (cDNA source: Clare Waterman, NIH; NM_009499); vascular epithelial cadherin (10), AgeI and NotI (human VE cadherin cDNA source: Origene; NM_001795.3), vimentin (7), BamHI and NotI (human vimentin cDNA source: Robert Goldman, Northwestern University; NM_003380.3), zyxin (6), BamHI and NotI (human zyxin cDNA source: Origene; NM_003461).

Article Title: Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction
Article Snippet: .. In brief, to construct plasmid pACLTR2 EF1α-hNRTN, the plasmid pACLTR2 EF1α were digested with BamHI filled in with T4 DNA polymerase and then digested with EcoRI to ligate ∼0.6 kb of hNRTN; pCMV6-XL5-hNRTN (Origene) was digested with Xba I and filled in with T4 DNA polymerase and digested with EcoRI to obtain the hNRTN cDNA. .. The recombinant vector AdLTR2 EF1α-hNRTN was produced by homologous recombination of pACLTR2EF1a-hNRTN with pJM17 in C7 cells.

Article Title: Pathogenic variants in the AFG3L2 proteolytic domain cause SCA28 through haploinsufficiency and proteostatic stress-driven OMA1 activation
Article Snippet: .. Complementary oligonucleotides (AFG3L2 FWD 5′ gatcgCTCCTCGTGCCTGGCGGCGTg 3′ and AFG3L2 REV 5′ aaaacACGCCGCCAGGCACGAGGAGc 3′) for gRNA were annealed and cloned into BamHI and BsmBI sites of pCas-Guide-EF1a-GFP CRISPR/Cas9 All-in-One vector (GE10018, OriGene). .. HEK 293 T cells were transfected with the pCas-Guide-EF1a-GFP vector containing the AFG3L2 targeting sequence, and 48 hours after transfection, Green Fluorescent Protein (GFP)-positive cells were sorted through fluorescence-activated cell sorting.

Expressing:

Article Title: Myosin 1b Regulates Amino Acid Transport by Associating Transporters with the Apical Plasma Membrane of Kidney Cells
Article Snippet: .. For collectrin expression, the GFP cassette of pLenti-CMV-GFP-Hygro (Addgene plasmid 17446; [ ]) was excised with BamHI and SalI and replaced with the cDNA of human collectrin (Tmem27 ) prepared by PCR from Tmem27-pCMV6-XL5 (SC122895; Origene, Rockville, MD) with the appropriate restriction sites. ..

Article Title: A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum
Article Snippet: Fusion Plasmid Construction mNeonGreen fluorescent protein expression vectors were constructed using C1 and N1 (Clontech-style) cloning vectors. .. To prepare mNeonGreen N-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (cDNA source, Tom Keller, Florida State University (FSU), Tallahassee, FL, U.S.A.; NM_001130005.1); human calnexin, AgeI and NotI (Origene; NM_001746.3); c-src (7), BamHI and EcoRI (chicken c-src cDNA source: Marilyn Resh, Sloan-Kettering, New York; XM_001232484.1); connexin-43 (7), BamHI and NotI (rat C×43 cDNA source: Matthias Falk, Lehigh U; NM_001004099.1); EB3 (7), BglII and BamHI (EB3 cDNA source: Lynne Cassimeris, Lehigh University; NM_012326.2); human keratin 18, EcoRI and NotI (Open Biosystems; NM_199187.1); lamin B1 (10), EcoRI and BamHI (human lamin B1 cDNA source: George Patterson, NIH; NM_005573.2); Lifeact (7), BamHI and NotI (Lifeact cDNA source: IDT); mouse mannosidase 2 (112 N-terminal amino acids, MANNII; 10), NheI and BamHI (cDNA source: Jennifer Lippincott-Schwartz, NIH; NM_008549.2); myosin IIA (14) NheI and BglII (mouse myosin IIA cDNA source: Origene; NM_022410.2); human nucleoporin 50kDa, BamHI and NotI (NUP50 cDNA source: Origene; NM_007172.2); human pyruvate dehydrogenase, AgeI and NotI (human PDHA1 cDNA source: Origene; NM_000284); human peroxisomal membrane protein, NotI and AgeI (PMP cDNA source: Origene; NM_018663.1); human MAP Tau (10), AgeI and NotI (MAP Tau cDNA source: Origene; NM_016841); human TfR (20), BamHI and NotI (transferrin receptor cDNA source: George Patterson, NIH; NM_NM_003234); human TPX2 (10), AgeI and NotI (TPX2 cDNA source: Patricia Wadsworth, University of Massachusetts, Amherst; NM_012112.4); mouse VASP (10), NheI and BamHI (cDNA source: Clare Waterman, NIH; NM_009499); vascular epithelial cadherin (10), AgeI and NotI (human VE cadherin cDNA source: Origene; NM_001795.3), vimentin (7), BamHI and NotI (human vimentin cDNA source: Robert Goldman, Northwestern University; NM_003380.3), zyxin (6), BamHI and NotI (human zyxin cDNA source: Origene; NM_003461).

Article Title: Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction
Article Snippet: The AdLTR2 EF1α-hNRTN vector used for these experiments, here abbreviated to AdNRTN for simplicity, is a hybrid E1-deleted serotype 5 recombinant adenoviral vector with a human NRTN cDNA (hNRTN; Origene Technologies, Rockville, MD, USA), which can persist long-term transgene expression in the salivary gland. .. In brief, to construct plasmid pACLTR2 EF1α-hNRTN, the plasmid pACLTR2 EF1α were digested with BamHI filled in with T4 DNA polymerase and then digested with EcoRI to ligate ∼0.6 kb of hNRTN; pCMV6-XL5-hNRTN (Origene) was digested with Xba I and filled in with T4 DNA polymerase and digested with EcoRI to obtain the hNRTN cDNA.

CRISPR:

Article Title: Pathogenic variants in the AFG3L2 proteolytic domain cause SCA28 through haploinsufficiency and proteostatic stress-driven OMA1 activation
Article Snippet: .. Complementary oligonucleotides (AFG3L2 FWD 5′ gatcgCTCCTCGTGCCTGGCGGCGTg 3′ and AFG3L2 REV 5′ aaaacACGCCGCCAGGCACGAGGAGc 3′) for gRNA were annealed and cloned into BamHI and BsmBI sites of pCas-Guide-EF1a-GFP CRISPR/Cas9 All-in-One vector (GE10018, OriGene). .. HEK 293 T cells were transfected with the pCas-Guide-EF1a-GFP vector containing the AFG3L2 targeting sequence, and 48 hours after transfection, Green Fluorescent Protein (GFP)-positive cells were sorted through fluorescence-activated cell sorting.

Sequencing:

Article Title: Pathogenic variants in the AFG3L2 proteolytic domain cause SCA28 through haploinsufficiency and proteostatic stress-driven OMA1 activation
Article Snippet: Generation of monoclonal AFG3L2+/− HEK 293 T cells by CRISPR/Cas9-based technology A 20 bp targeting sequence for spCas9 was selected in the coding sequence of exon 1 of AFG3L2 by using NCBI BLAST ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). .. Complementary oligonucleotides (AFG3L2 FWD 5′ gatcgCTCCTCGTGCCTGGCGGCGTg 3′ and AFG3L2 REV 5′ aaaacACGCCGCCAGGCACGAGGAGc 3′) for gRNA were annealed and cloned into BamHI and BsmBI sites of pCas-Guide-EF1a-GFP CRISPR/Cas9 All-in-One vector (GE10018, OriGene).

FACS:

Article Title: Pathogenic variants in the AFG3L2 proteolytic domain cause SCA28 through haploinsufficiency and proteostatic stress-driven OMA1 activation
Article Snippet: Complementary oligonucleotides (AFG3L2 FWD 5′ gatcgCTCCTCGTGCCTGGCGGCGTg 3′ and AFG3L2 REV 5′ aaaacACGCCGCCAGGCACGAGGAGc 3′) for gRNA were annealed and cloned into BamHI and BsmBI sites of pCas-Guide-EF1a-GFP CRISPR/Cas9 All-in-One vector (GE10018, OriGene). .. HEK 293 T cells were transfected with the pCas-Guide-EF1a-GFP vector containing the AFG3L2 targeting sequence, and 48 hours after transfection, Green Fluorescent Protein (GFP)-positive cells were sorted through fluorescence-activated cell sorting.

Real-time Polymerase Chain Reaction:

Article Title: Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction
Article Snippet: In brief, to construct plasmid pACLTR2 EF1α-hNRTN, the plasmid pACLTR2 EF1α were digested with BamHI filled in with T4 DNA polymerase and then digested with EcoRI to ligate ∼0.6 kb of hNRTN; pCMV6-XL5-hNRTN (Origene) was digested with Xba I and filled in with T4 DNA polymerase and digested with EcoRI to obtain the hNRTN cDNA. .. The titers (particles/mL) of purified vectors were determined by qPCR using primers from the E2 region.

Recombinant:

Article Title: Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction
Article Snippet: The AdLTR2 EF1α-hNRTN vector used for these experiments, here abbreviated to AdNRTN for simplicity, is a hybrid E1-deleted serotype 5 recombinant adenoviral vector with a human NRTN cDNA (hNRTN; Origene Technologies, Rockville, MD, USA), which can persist long-term transgene expression in the salivary gland. .. In brief, to construct plasmid pACLTR2 EF1α-hNRTN, the plasmid pACLTR2 EF1α were digested with BamHI filled in with T4 DNA polymerase and then digested with EcoRI to ligate ∼0.6 kb of hNRTN; pCMV6-XL5-hNRTN (Origene) was digested with Xba I and filled in with T4 DNA polymerase and digested with EcoRI to obtain the hNRTN cDNA.

Homologous Recombination:

Article Title: Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction
Article Snippet: In brief, to construct plasmid pACLTR2 EF1α-hNRTN, the plasmid pACLTR2 EF1α were digested with BamHI filled in with T4 DNA polymerase and then digested with EcoRI to ligate ∼0.6 kb of hNRTN; pCMV6-XL5-hNRTN (Origene) was digested with Xba I and filled in with T4 DNA polymerase and digested with EcoRI to obtain the hNRTN cDNA. .. The recombinant vector AdLTR2 EF1α-hNRTN was produced by homologous recombination of pACLTR2EF1a-hNRTN with pJM17 in C7 cells.

Software:

Article Title: Pathogenic variants in the AFG3L2 proteolytic domain cause SCA28 through haploinsufficiency and proteostatic stress-driven OMA1 activation
Article Snippet: Complementary oligonucleotides (AFG3L2 FWD 5′ gatcgCTCCTCGTGCCTGGCGGCGTg 3′ and AFG3L2 REV 5′ aaaacACGCCGCCAGGCACGAGGAGc 3′) for gRNA were annealed and cloned into BamHI and BsmBI sites of pCas-Guide-EF1a-GFP CRISPR/Cas9 All-in-One vector (GE10018, OriGene). .. Sorting was performed with FACSAria Fusion instrument and FACSDiva 8.0 software (BD).

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