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Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either <t>BamHI</t> or <t>EcoRI.</t> M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.
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1) Product Images from "Design and Characterization of Bioengineered Cancer-Like Stem Cells"

Article Title: Design and Characterization of Bioengineered Cancer-Like Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0141172

Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.
Figure Legend Snippet: Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.

Techniques Used: Subcloning, Clone Assay, DNA Sequencing

Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.
Figure Legend Snippet: Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.

Techniques Used: Subcloning, Clone Assay, DNA Sequencing

Related Articles

Clone Assay:

Article Title: The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner
Article Snippet: .. PCR products were cloned into pMiniT (NEB) and then subcloned into miniCTX lacZ using the restriction enzymes HindIII and BamHI, HindIII and EcoRI, or KpnI and BamHI (NEB). ..

Article Title: Distinct Functions of Bombyx mori Peptidoglycan Recognition Protein 2 in Immune Responses to Bacteria and Viruses
Article Snippet: .. BmPGRP2 Localization, PGN Treatment, RNA Interference (RNAi), and BmPGRP2 Overexpression A synthetic sequence includes B. mori A4 promoter (A4P) and GFP was cloned into the empty vector 1180 (GenBank: U13865.1 ) using SalI (#R3138V, NEB, USA) and BamHI (#R3136V, NEB, USA) restriction enzymes, followed adding BmPGRP2-1 or -2 [using BamHI and NotI (#R3189V, NEB, USA)] and Simian virus (SV)40 [using NotI and HindIII (#R3104V, NEB, USA)] to construct 1180-A4P-GFP-BmPGRP2-1 /2-SV40, which was transfected into BmE cells for subcellular localization analysis. .. PGN-EB (PGN from E. coli 0111:B4, #tlrl-pgneb), PGN-BS (PGN from Bacillus subtilis , #tlrl-pgnbs), PGN-SA (PGN from Staphylococcus aureus , #tlrl-pgnsa), and LPS-EB (LPS from E. coli 0111:B4, #tlrl-eblps) were purchased from InvivoGen (San Diego, CA, USA) and added to BmE cells.

Transfection:

Article Title: Distinct Functions of Bombyx mori Peptidoglycan Recognition Protein 2 in Immune Responses to Bacteria and Viruses
Article Snippet: .. BmPGRP2 Localization, PGN Treatment, RNA Interference (RNAi), and BmPGRP2 Overexpression A synthetic sequence includes B. mori A4 promoter (A4P) and GFP was cloned into the empty vector 1180 (GenBank: U13865.1 ) using SalI (#R3138V, NEB, USA) and BamHI (#R3136V, NEB, USA) restriction enzymes, followed adding BmPGRP2-1 or -2 [using BamHI and NotI (#R3189V, NEB, USA)] and Simian virus (SV)40 [using NotI and HindIII (#R3104V, NEB, USA)] to construct 1180-A4P-GFP-BmPGRP2-1 /2-SV40, which was transfected into BmE cells for subcellular localization analysis. .. PGN-EB (PGN from E. coli 0111:B4, #tlrl-pgneb), PGN-BS (PGN from Bacillus subtilis , #tlrl-pgnbs), PGN-SA (PGN from Staphylococcus aureus , #tlrl-pgnsa), and LPS-EB (LPS from E. coli 0111:B4, #tlrl-eblps) were purchased from InvivoGen (San Diego, CA, USA) and added to BmE cells.

Ligation:

Article Title: Design and Characterization of Bioengineered Cancer-Like Stem Cells
Article Snippet: Sub-Cloning of Genes to pMSCV To perform sub-cloning, Hras V12 and SV40 large T antigene (LTg) were separated from pBABE-Hras V12 and pBABE-SV40 LTg by the enzymatic digestion with BamHI and EcoRI (NEB, Ipswich, MA), or with BamHI (NEB), respectively. .. Integration of Hras V12 and SV40LTg into either pMSCV-GFP or pMSCV-RFP was performed by ligation with T4-ligase (NEB) and produced pMSCV-Hras V12-GFP and pMSCV-SV40 LTg-RFP.

Radial Immuno Diffusion:

Article Title: Distinct Functions of Bombyx mori Peptidoglycan Recognition Protein 2 in Immune Responses to Bacteria and Viruses
Article Snippet: BmPGRP2 Localization, PGN Treatment, RNA Interference (RNAi), and BmPGRP2 Overexpression A synthetic sequence includes B. mori A4 promoter (A4P) and GFP was cloned into the empty vector 1180 (GenBank: U13865.1 ) using SalI (#R3138V, NEB, USA) and BamHI (#R3136V, NEB, USA) restriction enzymes, followed adding BmPGRP2-1 or -2 [using BamHI and NotI (#R3189V, NEB, USA)] and Simian virus (SV)40 [using NotI and HindIII (#R3104V, NEB, USA)] to construct 1180-A4P-GFP-BmPGRP2-1 /2-SV40, which was transfected into BmE cells for subcellular localization analysis. .. Total RNA was extracted 0, 6, 12, and 24 h after treatment to detect the presence of BmPGRP2-1, imd , and the AMP gene attacin (att )2 .

Subcloning:

Article Title: Design and Characterization of Bioengineered Cancer-Like Stem Cells
Article Snippet: .. Sub-Cloning of Genes to pMSCV To perform sub-cloning, Hras V12 and SV40 large T antigene (LTg) were separated from pBABE-Hras V12 and pBABE-SV40 LTg by the enzymatic digestion with BamHI and EcoRI (NEB, Ipswich, MA), or with BamHI (NEB), respectively. .. Integration of Hras V12 and SV40LTg into either pMSCV-GFP or pMSCV-RFP was performed by ligation with T4-ligase (NEB) and produced pMSCV-Hras V12-GFP and pMSCV-SV40 LTg-RFP.

Construct:

Article Title: The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner
Article Snippet: PCR products were cloned into pMiniT (NEB) and then subcloned into miniCTX lacZ using the restriction enzymes HindIII and BamHI, HindIII and EcoRI, or KpnI and BamHI (NEB). .. To construct rsmY and rsmZ transcriptional fusions, the rsmYTFFEcoRI/rsmYTFR and rsmZTFF/rsmZTFR primer pairs, respectively, were used.

Article Title: Distinct Functions of Bombyx mori Peptidoglycan Recognition Protein 2 in Immune Responses to Bacteria and Viruses
Article Snippet: .. BmPGRP2 Localization, PGN Treatment, RNA Interference (RNAi), and BmPGRP2 Overexpression A synthetic sequence includes B. mori A4 promoter (A4P) and GFP was cloned into the empty vector 1180 (GenBank: U13865.1 ) using SalI (#R3138V, NEB, USA) and BamHI (#R3136V, NEB, USA) restriction enzymes, followed adding BmPGRP2-1 or -2 [using BamHI and NotI (#R3189V, NEB, USA)] and Simian virus (SV)40 [using NotI and HindIII (#R3104V, NEB, USA)] to construct 1180-A4P-GFP-BmPGRP2-1 /2-SV40, which was transfected into BmE cells for subcellular localization analysis. .. PGN-EB (PGN from E. coli 0111:B4, #tlrl-pgneb), PGN-BS (PGN from Bacillus subtilis , #tlrl-pgnbs), PGN-SA (PGN from Staphylococcus aureus , #tlrl-pgnsa), and LPS-EB (LPS from E. coli 0111:B4, #tlrl-eblps) were purchased from InvivoGen (San Diego, CA, USA) and added to BmE cells.

Purification:

Article Title: The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner
Article Snippet: PCR products were cloned into pMiniT (NEB) and then subcloned into miniCTX lacZ using the restriction enzymes HindIII and BamHI, HindIII and EcoRI, or KpnI and BamHI (NEB). .. PCR products were purified, cut with restriction enzymes, and inserted into the EcoRI and BamHI sites of miniCTX lacZ using T4 DNA ligase (NEB).

Produced:

Article Title: Design and Characterization of Bioengineered Cancer-Like Stem Cells
Article Snippet: Sub-Cloning of Genes to pMSCV To perform sub-cloning, Hras V12 and SV40 large T antigene (LTg) were separated from pBABE-Hras V12 and pBABE-SV40 LTg by the enzymatic digestion with BamHI and EcoRI (NEB, Ipswich, MA), or with BamHI (NEB), respectively. .. Integration of Hras V12 and SV40LTg into either pMSCV-GFP or pMSCV-RFP was performed by ligation with T4-ligase (NEB) and produced pMSCV-Hras V12-GFP and pMSCV-SV40 LTg-RFP.

Polymerase Chain Reaction:

Article Title: The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner
Article Snippet: .. PCR products were cloned into pMiniT (NEB) and then subcloned into miniCTX lacZ using the restriction enzymes HindIII and BamHI, HindIII and EcoRI, or KpnI and BamHI (NEB). ..

Generated:

Article Title: The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner
Article Snippet: Upstream DNA fragments containing promoter regions were generated by using primers listed in in conjunction with Q5 polymerase (New England BioLabs). .. PCR products were cloned into pMiniT (NEB) and then subcloned into miniCTX lacZ using the restriction enzymes HindIII and BamHI, HindIII and EcoRI, or KpnI and BamHI (NEB).

Infection:

Article Title: Distinct Functions of Bombyx mori Peptidoglycan Recognition Protein 2 in Immune Responses to Bacteria and Viruses
Article Snippet: BmPGRP2 Localization, PGN Treatment, RNA Interference (RNAi), and BmPGRP2 Overexpression A synthetic sequence includes B. mori A4 promoter (A4P) and GFP was cloned into the empty vector 1180 (GenBank: U13865.1 ) using SalI (#R3138V, NEB, USA) and BamHI (#R3136V, NEB, USA) restriction enzymes, followed adding BmPGRP2-1 or -2 [using BamHI and NotI (#R3189V, NEB, USA)] and Simian virus (SV)40 [using NotI and HindIII (#R3104V, NEB, USA)] to construct 1180-A4P-GFP-BmPGRP2-1 /2-SV40, which was transfected into BmE cells for subcellular localization analysis. .. Fifth instar silkworms were orally infected with E. coli and S. marcescens at 109 /larva, and extracted RNA was tested for the presence of BmPGRP2-1/2 and the AMP gene gloverin (glv )2 .

Sequencing:

Article Title: Distinct Functions of Bombyx mori Peptidoglycan Recognition Protein 2 in Immune Responses to Bacteria and Viruses
Article Snippet: .. BmPGRP2 Localization, PGN Treatment, RNA Interference (RNAi), and BmPGRP2 Overexpression A synthetic sequence includes B. mori A4 promoter (A4P) and GFP was cloned into the empty vector 1180 (GenBank: U13865.1 ) using SalI (#R3138V, NEB, USA) and BamHI (#R3136V, NEB, USA) restriction enzymes, followed adding BmPGRP2-1 or -2 [using BamHI and NotI (#R3189V, NEB, USA)] and Simian virus (SV)40 [using NotI and HindIII (#R3104V, NEB, USA)] to construct 1180-A4P-GFP-BmPGRP2-1 /2-SV40, which was transfected into BmE cells for subcellular localization analysis. .. PGN-EB (PGN from E. coli 0111:B4, #tlrl-pgneb), PGN-BS (PGN from Bacillus subtilis , #tlrl-pgnbs), PGN-SA (PGN from Staphylococcus aureus , #tlrl-pgnsa), and LPS-EB (LPS from E. coli 0111:B4, #tlrl-eblps) were purchased from InvivoGen (San Diego, CA, USA) and added to BmE cells.

Over Expression:

Article Title: Distinct Functions of Bombyx mori Peptidoglycan Recognition Protein 2 in Immune Responses to Bacteria and Viruses
Article Snippet: .. BmPGRP2 Localization, PGN Treatment, RNA Interference (RNAi), and BmPGRP2 Overexpression A synthetic sequence includes B. mori A4 promoter (A4P) and GFP was cloned into the empty vector 1180 (GenBank: U13865.1 ) using SalI (#R3138V, NEB, USA) and BamHI (#R3136V, NEB, USA) restriction enzymes, followed adding BmPGRP2-1 or -2 [using BamHI and NotI (#R3189V, NEB, USA)] and Simian virus (SV)40 [using NotI and HindIII (#R3104V, NEB, USA)] to construct 1180-A4P-GFP-BmPGRP2-1 /2-SV40, which was transfected into BmE cells for subcellular localization analysis. .. PGN-EB (PGN from E. coli 0111:B4, #tlrl-pgneb), PGN-BS (PGN from Bacillus subtilis , #tlrl-pgnbs), PGN-SA (PGN from Staphylococcus aureus , #tlrl-pgnsa), and LPS-EB (LPS from E. coli 0111:B4, #tlrl-eblps) were purchased from InvivoGen (San Diego, CA, USA) and added to BmE cells.

Plasmid Preparation:

Article Title: Distinct Functions of Bombyx mori Peptidoglycan Recognition Protein 2 in Immune Responses to Bacteria and Viruses
Article Snippet: .. BmPGRP2 Localization, PGN Treatment, RNA Interference (RNAi), and BmPGRP2 Overexpression A synthetic sequence includes B. mori A4 promoter (A4P) and GFP was cloned into the empty vector 1180 (GenBank: U13865.1 ) using SalI (#R3138V, NEB, USA) and BamHI (#R3136V, NEB, USA) restriction enzymes, followed adding BmPGRP2-1 or -2 [using BamHI and NotI (#R3189V, NEB, USA)] and Simian virus (SV)40 [using NotI and HindIII (#R3104V, NEB, USA)] to construct 1180-A4P-GFP-BmPGRP2-1 /2-SV40, which was transfected into BmE cells for subcellular localization analysis. .. PGN-EB (PGN from E. coli 0111:B4, #tlrl-pgneb), PGN-BS (PGN from Bacillus subtilis , #tlrl-pgnbs), PGN-SA (PGN from Staphylococcus aureus , #tlrl-pgnsa), and LPS-EB (LPS from E. coli 0111:B4, #tlrl-eblps) were purchased from InvivoGen (San Diego, CA, USA) and added to BmE cells.

DNA Sequencing:

Article Title: Design and Characterization of Bioengineered Cancer-Like Stem Cells
Article Snippet: Sub-Cloning of Genes to pMSCV To perform sub-cloning, Hras V12 and SV40 large T antigene (LTg) were separated from pBABE-Hras V12 and pBABE-SV40 LTg by the enzymatic digestion with BamHI and EcoRI (NEB, Ipswich, MA), or with BamHI (NEB), respectively. .. Integration of the inserts was verified by both enzymatic digestion and DNA sequencing.

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    New England Biolabs bamhi hf
    Bamhi Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bamhi hf/product/New England Biolabs
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