r3101l  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    Name:
    EcoRI HF
    Description:
    EcoRI HF 50 000 units
    Catalog Number:
    r3101l
    Price:
    249
    Size:
    50 000 units
    Category:
    Restriction Enzymes
    Buy from Supplier


    Structured Review

    New England Biolabs r3101l
    EcoRI HF
    EcoRI HF 50 000 units
    https://www.bioz.com/result/r3101l/product/New England Biolabs
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    r3101l - by Bioz Stars, 2020-02
    95/100 stars

    Related Products / Commonly Used Together

    r3552l
    t4 dna ligase

    Images

    Related Articles

    Clone Assay:

    Article Title: Synapse-to-Nucleus Communication through NFAT Is Mediated by L-type Ca2+ Channel Ca2+ Spike Propagation to the Soma
    Article Snippet: The mNFATc4 insert and pSGFP2-C1 backbone were digested with XhoI and EcoRI-HF (NEB) in dual restriction digests, gel purified and ligated with T4 DNA ligase (NEB). .. The NFATc3 cloning fragment used previously and the ΔHindIII mEos3.2-C1 were then digested with HindIII and ApaI, gel purified and ligated.

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: All the standard parts were domesticated for Aar I and Bsa I when necessary and were cloned into mUAV. .. Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel).

    Amplification:

    Article Title: Synapse-to-Nucleus Communication through NFAT Is Mediated by L-type Ca2+ Channel Ca2+ Spike Propagation to the Soma
    Article Snippet: To generate the sGFP-NFATc4 construct, mNFATc4 ( ) was PCR amplified with primers (forward 5′-CGACTCGA GGAGGGGCCGCAAGCTGCG-3′ and reverse 5′-CGATGAATTCTCAGGCAGGAGGCTCTTCTCC-3′) to introduce a 5′ XhoI site and a 3′ EcoRI site to the resulting product, which was then gel purified. .. The mNFATc4 insert and pSGFP2-C1 backbone were digested with XhoI and EcoRI-HF (NEB) in dual restriction digests, gel purified and ligated with T4 DNA ligase (NEB).

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: They were then amplified using Q5 DNA polymerase (NEB) with primers bearing EcoRI , AarI , BsaI , and PstI restriction sites and 4 bp overhangs. .. Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel).

    DNA Ligation:

    Article Title: Calpain 5 Is Highly Expressed in the Central Nervous System (CNS), Carries Dual Nuclear Localization Signals, and Is Associated with Nuclear Promyelocytic Leukemia Protein Bodies *
    Article Snippet: EcoRI-HF and BamHI-HF were purchased from New England Biolabs, Ipswich, MA. .. The Rapid DNA ligation kit (11-635-379-001) was obtained from Roche Applied Science.

    Synthesized:

    Article Title: Effects of claudin-1 downregulation on the physiological processes of gallbladder cancer SGC996 cells
    Article Snippet: The negative-control cells were transfected with the virus CON077 synthesized by Shanghai GeneChem Co., Ltd. .. R3552L and R3101L, respectively) and T4 DNA ligase used for plasmid construction were purchased from New England BioLabs, Inc. (Ipswich, MA, USA).

    Article Title: Regulatory Protein BBD18 of the Lyme Disease Spirochete: Essential Role During Tick Acquisition?
    Article Snippet: Variant bbd18 genes were synthesized by GenScript Corporation (Piscataway, NJ) with EcoRI recognition sites at both ends. .. These synthetic genes were excised from pUC57 with EcoRI-HF (New England Biolabs, Ipswich, MA) and ligated into appropriately digested and dephosphorylated pBSV2*flaBp ( ) in a manner following the construction of pBSV2*flaBp-bbd18, which contains the wild-type bbd18 gene under expression of the constitutive flaB promoter ( ).

    Construct:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: .. The double-stranded product was then purified using the Zymo Research DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA) and subjected to EcoRI/BamHI restriction digest using BamHI-HF (New England Biolabs) and EcoRI-HF (New England Biolabs) to prepare the construct for ligation into an EcoRI/BamHI-digested pUC19 backbone. .. Digested vector and construct were run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA), and the appropriate bands were manually excised.

    Article Title: Synapse-to-Nucleus Communication through NFAT Is Mediated by L-type Ca2+ Channel Ca2+ Spike Propagation to the Soma
    Article Snippet: Paragraph title: Plasmid DNA constructs ... The mNFATc4 insert and pSGFP2-C1 backbone were digested with XhoI and EcoRI-HF (NEB) in dual restriction digests, gel purified and ligated with T4 DNA ligase (NEB).

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel). .. Ligation was mediated by T7 DNA ligase (NEB) to construct mUAV, Level 1 and Level 2 Acceptor Vectors.

    Article Title: Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador, et al. Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador
    Article Snippet: 2.3 ddRADseq library generation and sequencing We constructed double‐digested restriction‐site‐associated DNA sequencing (ddRAD) libraries on a subset of 125 samples of O. sylvatica following the protocol in Peterson, Weber, Kay, Fisher, and Hoekstra ( ). .. Genomic DNA of each sample (1 μg) was digested using 1 μl of EcoRI‐HF (20,000 U/ml) and 1 μl of SphI‐HF (20,000 U/ml) (New England Biolabs, Ipswitch, MA, USA) following the manufacturer's protocol.

    Electrophoresis:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: The double-stranded product was then purified using the Zymo Research DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA) and subjected to EcoRI/BamHI restriction digest using BamHI-HF (New England Biolabs) and EcoRI-HF (New England Biolabs) to prepare the construct for ligation into an EcoRI/BamHI-digested pUC19 backbone. .. Digested vector and construct were run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA), and the appropriate bands were manually excised.

    Incubation:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: The double-stranded product was then purified using the Zymo Research DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA) and subjected to EcoRI/BamHI restriction digest using BamHI-HF (New England Biolabs) and EcoRI-HF (New England Biolabs) to prepare the construct for ligation into an EcoRI/BamHI-digested pUC19 backbone. .. Ligation reactions were performed with high-concentration T4 DNA ligase (Life Technologies) with a 1:6 vector to insert ratio, followed by overnight incubation at 16°C.

    Cell Culture:

    Article Title: Effects of claudin-1 downregulation on the physiological processes of gallbladder cancer SGC996 cells
    Article Snippet: R3552L and R3101L, respectively) and T4 DNA ligase used for plasmid construction were purchased from New England BioLabs, Inc. (Ipswich, MA, USA). .. Following trypsin digestion (Sangon Biotech Co., Ltd., Shanghai, China), all cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin G and 100 µg/ml streptomycin.

    Expressing:

    Article Title: Effects of claudin-1 downregulation on the physiological processes of gallbladder cancer SGC996 cells
    Article Snippet: SGC996 cells in the logarithmic growth phase were transfected with claudin-1-RNA interference lentivirus (LV-CLDN1-RNAi) (Shanghai GeneChem Co., Ltd.) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to suppress claudin-1 expression. .. R3552L and R3101L, respectively) and T4 DNA ligase used for plasmid construction were purchased from New England BioLabs, Inc. (Ipswich, MA, USA).

    Article Title: Regulatory Protein BBD18 of the Lyme Disease Spirochete: Essential Role During Tick Acquisition?
    Article Snippet: .. These synthetic genes were excised from pUC57 with EcoRI-HF (New England Biolabs, Ipswich, MA) and ligated into appropriately digested and dephosphorylated pBSV2*flaBp ( ) in a manner following the construction of pBSV2*flaBp-bbd18, which contains the wild-type bbd18 gene under expression of the constitutive flaB promoter ( ). ..

    Genome Wide:

    Article Title: Beyond the Coral Triangle: high genetic diversity and near panmixia in Singapore's populations of the broadcast spawning sea star Protoreaster nodosus
    Article Snippet: A double-digest restriction enzyme associated DNA sequencing (ddRADseq) library was then prepared from these extracts for genome-wide SNP analyses, using the adapters and PCR primer pairs in Peterson et al . .. A total of 100 ng DNA from each sample was simultaneously double-digested with restriction enzymes and ligated to adapters in duplicate 13 µl reactions at 37°C for 3.5 h. Each reaction contained 5 U EcoRI-HF® (NEB), 1 U MspI, 80 U T4 DNA ligase, 1× T4 DNA ligase buffer, 50 mM NaCl, 0.05 mg ml−1 bovine serum albumin and 3.85 µM of each adapter.

    Transformation Assay:

    Article Title: Regulatory Protein BBD18 of the Lyme Disease Spirochete: Essential Role During Tick Acquisition?
    Article Snippet: These synthetic genes were excised from pUC57 with EcoRI-HF (New England Biolabs, Ipswich, MA) and ligated into appropriately digested and dephosphorylated pBSV2*flaBp ( ) in a manner following the construction of pBSV2*flaBp-bbd18, which contains the wild-type bbd18 gene under expression of the constitutive flaB promoter ( ). .. These shuttle vectors were transformed into B312 and assessed by immunoblotting for their ability to suppress OspC synthesis.

    Article Title: The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri
    Article Snippet: Approximately 3-μ g genomic DNA was digested by EcoRI-HF (New England Biolabs, Ipswich, MA) in a 30-μ L reaction at 37°C. .. The DNA was self-ligated using T4 DNA ligase (New England Biolabs), transformed by electroporation into EC100Dpir+ (Epicentre Biotechnologies), and selected on BHI containing 150 μ g mL−1 erythromycin.

    Conjugation Assay:

    Article Title: The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri
    Article Snippet: Transposon mutagenesis screen The reporter plasmid pTM268 was introduced by conjugation into a Tn5 transposon-mutant library of ES114 that has been previously described (Miyashiro et al. ). .. Approximately 3-μ g genomic DNA was digested by EcoRI-HF (New England Biolabs, Ipswich, MA) in a 30-μ L reaction at 37°C.

    Transfection:

    Article Title: Effects of claudin-1 downregulation on the physiological processes of gallbladder cancer SGC996 cells
    Article Snippet: The negative-control cells were transfected with the virus CON077 synthesized by Shanghai GeneChem Co., Ltd. .. R3552L and R3101L, respectively) and T4 DNA ligase used for plasmid construction were purchased from New England BioLabs, Inc. (Ipswich, MA, USA).

    Ligation:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: .. The double-stranded product was then purified using the Zymo Research DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA) and subjected to EcoRI/BamHI restriction digest using BamHI-HF (New England Biolabs) and EcoRI-HF (New England Biolabs) to prepare the construct for ligation into an EcoRI/BamHI-digested pUC19 backbone. .. Digested vector and construct were run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA), and the appropriate bands were manually excised.

    Article Title: Quantitative assessment of LASSO probe assembly and long-read multiplexed cloning
    Article Snippet: LASSO probe assembly The LASSO probe assembly protocol is the same as that previously described [ ], with exceptions during the ligation protocol (self-circularization). .. Approximately 45 μl of solution containing gel-purified fusion PCR product as described were digested by adding 5 μl of CutSmart 10X buffer (NEB) and 1 μl (20 units/μl) of EcoRI-HF (NEB) for 1 h at 37 °C followed by a denaturation step for 10 min at 80 °C.

    Article Title: Utilizing field collected insects for next generation sequencing: Effects of sampling, storage, and DNA extraction methods. Utilizing field collected insects for next generation sequencing: Effects of sampling, storage, and DNA extraction methods
    Article Snippet: 2.3 Library preparation and ddRAD sequencing One hundred ng of DNA per sample after normalization using PicoGreen® measurement were digested in 1X NEB Cut Smart Buffer and 100 U each EcoRI‐HF and MspI (NEB), for a final volume of 25 µl, at 37°C for 4 hr. .. Following a 20 min 80°C enzyme inactivation, samples were held at 12°C until ligation.

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel). .. Ligation was mediated by T7 DNA ligase (NEB) to construct mUAV, Level 1 and Level 2 Acceptor Vectors.

    Article Title: Double‐digest RADseq loci using standard Illumina indexes improve deep and shallow phylogenetic resolution of Lophodermium, a widespread fungal endophyte of pine needles. Double‐digest RADseq loci using standard Illumina indexes improve deep and shallow phylogenetic resolution of Lophodermium, a widespread fungal endophyte of pine needles
    Article Snippet: Three to nine hundred nanograms of DNA per sample (6 μl total; 50–150 ng/μl) was double‐digested using the rare‐cutting EcoRI‐HF and the frequent‐cutting MseI enzymes (New England BioLabs, Ipswich, MA). .. In cases where starting DNA concentrations were low, duplicate samples underwent digestion and ligation steps and then were pooled (see detailed workbench protocol for additional tips and similarities with Kess et al. ( ) in Appendices S1–S2).

    Introduce:

    Article Title: Synapse-to-Nucleus Communication through NFAT Is Mediated by L-type Ca2+ Channel Ca2+ Spike Propagation to the Soma
    Article Snippet: To generate the sGFP-NFATc4 construct, mNFATc4 ( ) was PCR amplified with primers (forward 5′-CGACTCGA GGAGGGGCCGCAAGCTGCG-3′ and reverse 5′-CGATGAATTCTCAGGCAGGAGGCTCTTCTCC-3′) to introduce a 5′ XhoI site and a 3′ EcoRI site to the resulting product, which was then gel purified. .. The mNFATc4 insert and pSGFP2-C1 backbone were digested with XhoI and EcoRI-HF (NEB) in dual restriction digests, gel purified and ligated with T4 DNA ligase (NEB).

    Digital PCR:

    Article Title: Linked read sequencing resolves complex genomic rearrangements in gastric cancer metastases
    Article Snippet: Paragraph title: Identifying FGFR2 copy number using droplet digital PCR ... Briefly, gDNA was first digested by EcoRI-HF (NEB) and cleaned up by AMPure XP beads (Beckman Coulter).

    DNA Sequencing:

    Article Title: Beyond the Coral Triangle: high genetic diversity and near panmixia in Singapore's populations of the broadcast spawning sea star Protoreaster nodosus
    Article Snippet: A double-digest restriction enzyme associated DNA sequencing (ddRADseq) library was then prepared from these extracts for genome-wide SNP analyses, using the adapters and PCR primer pairs in Peterson et al . .. A total of 100 ng DNA from each sample was simultaneously double-digested with restriction enzymes and ligated to adapters in duplicate 13 µl reactions at 37°C for 3.5 h. Each reaction contained 5 U EcoRI-HF® (NEB), 1 U MspI, 80 U T4 DNA ligase, 1× T4 DNA ligase buffer, 50 mM NaCl, 0.05 mg ml−1 bovine serum albumin and 3.85 µM of each adapter.

    Article Title: Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador, et al. Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador
    Article Snippet: 2.3 ddRADseq library generation and sequencing We constructed double‐digested restriction‐site‐associated DNA sequencing (ddRAD) libraries on a subset of 125 samples of O. sylvatica following the protocol in Peterson, Weber, Kay, Fisher, and Hoekstra ( ). .. Genomic DNA of each sample (1 μg) was digested using 1 μl of EcoRI‐HF (20,000 U/ml) and 1 μl of SphI‐HF (20,000 U/ml) (New England Biolabs, Ipswitch, MA, USA) following the manufacturer's protocol.

    Sequencing:

    Article Title: Synapse-to-Nucleus Communication through NFAT Is Mediated by L-type Ca2+ Channel Ca2+ Spike Propagation to the Soma
    Article Snippet: The mNFATc4 insert and pSGFP2-C1 backbone were digested with XhoI and EcoRI-HF (NEB) in dual restriction digests, gel purified and ligated with T4 DNA ligase (NEB). .. To generate the mEos3.2-NFATc3 construct, the HindIII restriction site within the mEos coding sequence of mEos3.2-C1 was deleted with a site-directed mutagenesis PCR reaction (forward 5′-GTATTCGTGGGAACGAAGTTTGA-3′; reverse 5′- CCGTCTTCGAAAGTCAAACTTCG-3′).

    Article Title: Utilizing field collected insects for next generation sequencing: Effects of sampling, storage, and DNA extraction methods. Utilizing field collected insects for next generation sequencing: Effects of sampling, storage, and DNA extraction methods
    Article Snippet: .. 2.3 Library preparation and ddRAD sequencing One hundred ng of DNA per sample after normalization using PicoGreen® measurement were digested in 1X NEB Cut Smart Buffer and 100 U each EcoRI‐HF and MspI (NEB), for a final volume of 25 µl, at 37°C for 4 hr. .. Following a 20 min 80°C enzyme inactivation, samples were held at 12°C until ligation.

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel). .. To construct the 50 bp linkers in the Auxiliary Plasmids, a short sequence was PCR amplified from scOrange gene using primers that contained the appropriate overhangs and the Aar I and Bsa I recognition sites.

    Article Title: Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador, et al. Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador
    Article Snippet: Paragraph title: ddRADseq library generation and sequencing ... Genomic DNA of each sample (1 μg) was digested using 1 μl of EcoRI‐HF (20,000 U/ml) and 1 μl of SphI‐HF (20,000 U/ml) (New England Biolabs, Ipswitch, MA, USA) following the manufacturer's protocol.

    DNA Extraction:

    Article Title: Beyond the Coral Triangle: high genetic diversity and near panmixia in Singapore's populations of the broadcast spawning sea star Protoreaster nodosus
    Article Snippet: 2.3. ddRADseq library preparation DNA extracts were prepared from two to four tube feet following the manufacturer's protocol of the Biospin tissue genomic DNA extraction kit. .. A total of 100 ng DNA from each sample was simultaneously double-digested with restriction enzymes and ligated to adapters in duplicate 13 µl reactions at 37°C for 3.5 h. Each reaction contained 5 U EcoRI-HF® (NEB), 1 U MspI, 80 U T4 DNA ligase, 1× T4 DNA ligase buffer, 50 mM NaCl, 0.05 mg ml−1 bovine serum albumin and 3.85 µM of each adapter.

    Nucleic Acid Electrophoresis:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: The double-stranded nature of the product was verified using a SmaI (New England BioLabs, Ipswich, MA, USA) restriction digest and gel electrophoresis. .. The double-stranded product was then purified using the Zymo Research DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA) and subjected to EcoRI/BamHI restriction digest using BamHI-HF (New England Biolabs) and EcoRI-HF (New England Biolabs) to prepare the construct for ligation into an EcoRI/BamHI-digested pUC19 backbone.

    Fluorescence:

    Article Title: The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri
    Article Snippet: The resulting colonies were screened for elevated Green Fluorescent Protein (GFP) levels using a Leica MZFLIII fluorescence dissecting microscope (Leica Microsystems, Wetzlar, Germany), equipped with a GFP2 filter set. .. Approximately 3-μ g genomic DNA was digested by EcoRI-HF (New England Biolabs, Ipswich, MA) in a 30-μ L reaction at 37°C.

    Electroporation:

    Article Title: The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri
    Article Snippet: Approximately 3-μ g genomic DNA was digested by EcoRI-HF (New England Biolabs, Ipswich, MA) in a 30-μ L reaction at 37°C. .. The DNA was self-ligated using T4 DNA ligase (New England Biolabs), transformed by electroporation into EC100Dpir+ (Epicentre Biotechnologies), and selected on BHI containing 150 μ g mL−1 erythromycin.

    Mutagenesis:

    Article Title: Synapse-to-Nucleus Communication through NFAT Is Mediated by L-type Ca2+ Channel Ca2+ Spike Propagation to the Soma
    Article Snippet: The mNFATc4 insert and pSGFP2-C1 backbone were digested with XhoI and EcoRI-HF (NEB) in dual restriction digests, gel purified and ligated with T4 DNA ligase (NEB). .. To generate the mEos3.2-NFATc3 construct, the HindIII restriction site within the mEos coding sequence of mEos3.2-C1 was deleted with a site-directed mutagenesis PCR reaction (forward 5′-GTATTCGTGGGAACGAAGTTTGA-3′; reverse 5′- CCGTCTTCGAAAGTCAAACTTCG-3′).

    Article Title: Regulatory Protein BBD18 of the Lyme Disease Spirochete: Essential Role During Tick Acquisition?
    Article Snippet: Paragraph title: Site-directed mutagenesis of BBD18. ... These synthetic genes were excised from pUC57 with EcoRI-HF (New England Biolabs, Ipswich, MA) and ligated into appropriately digested and dephosphorylated pBSV2*flaBp ( ) in a manner following the construction of pBSV2*flaBp-bbd18, which contains the wild-type bbd18 gene under expression of the constitutive flaB promoter ( ).

    Article Title: The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri
    Article Snippet: Paragraph title: Transposon mutagenesis screen ... Approximately 3-μ g genomic DNA was digested by EcoRI-HF (New England Biolabs, Ipswich, MA) in a 30-μ L reaction at 37°C.

    Microscopy:

    Article Title: The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri
    Article Snippet: The resulting colonies were screened for elevated Green Fluorescent Protein (GFP) levels using a Leica MZFLIII fluorescence dissecting microscope (Leica Microsystems, Wetzlar, Germany), equipped with a GFP2 filter set. .. Approximately 3-μ g genomic DNA was digested by EcoRI-HF (New England Biolabs, Ipswich, MA) in a 30-μ L reaction at 37°C.

    Purification:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: .. The double-stranded product was then purified using the Zymo Research DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA) and subjected to EcoRI/BamHI restriction digest using BamHI-HF (New England Biolabs) and EcoRI-HF (New England Biolabs) to prepare the construct for ligation into an EcoRI/BamHI-digested pUC19 backbone. .. Digested vector and construct were run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA), and the appropriate bands were manually excised.

    Article Title: Synapse-to-Nucleus Communication through NFAT Is Mediated by L-type Ca2+ Channel Ca2+ Spike Propagation to the Soma
    Article Snippet: .. The mNFATc4 insert and pSGFP2-C1 backbone were digested with XhoI and EcoRI-HF (NEB) in dual restriction digests, gel purified and ligated with T4 DNA ligase (NEB). .. To generate the mEos3.2-NFATc3 construct, the HindIII restriction site within the mEos coding sequence of mEos3.2-C1 was deleted with a site-directed mutagenesis PCR reaction (forward 5′-GTATTCGTGGGAACGAAGTTTGA-3′; reverse 5′- CCGTCTTCGAAAGTCAAACTTCG-3′).

    Article Title: Quantitative assessment of LASSO probe assembly and long-read multiplexed cloning
    Article Snippet: Approximately 45 μl of solution containing gel-purified fusion PCR product as described were digested by adding 5 μl of CutSmart 10X buffer (NEB) and 1 μl (20 units/μl) of EcoRI-HF (NEB) for 1 h at 37 °C followed by a denaturation step for 10 min at 80 °C. .. EcoRI digested DNA was purified with Agencourt AMPure XP beads (Beckman Coulter) and eluted in 40 μl ddH2 O.

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: .. Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel). .. Ligation was mediated by T7 DNA ligase (NEB) to construct mUAV, Level 1 and Level 2 Acceptor Vectors.

    Article Title: Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador, et al. Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador
    Article Snippet: Genomic DNA of each sample (1 μg) was digested using 1 μl of EcoRI‐HF (20,000 U/ml) and 1 μl of SphI‐HF (20,000 U/ml) (New England Biolabs, Ipswitch, MA, USA) following the manufacturer's protocol. .. Purified digested DNA (100 ng) was ligated to double‐stranded adapters (biotin‐labeled on P2 adapter) with a unique inline barcode using T4 DNA ligase (New England Biolabs) and purified with Agencourt Ampure XP beads.

    Polymerase Chain Reaction:

    Article Title: Beyond the Coral Triangle: high genetic diversity and near panmixia in Singapore's populations of the broadcast spawning sea star Protoreaster nodosus
    Article Snippet: A double-digest restriction enzyme associated DNA sequencing (ddRADseq) library was then prepared from these extracts for genome-wide SNP analyses, using the adapters and PCR primer pairs in Peterson et al . .. A total of 100 ng DNA from each sample was simultaneously double-digested with restriction enzymes and ligated to adapters in duplicate 13 µl reactions at 37°C for 3.5 h. Each reaction contained 5 U EcoRI-HF® (NEB), 1 U MspI, 80 U T4 DNA ligase, 1× T4 DNA ligase buffer, 50 mM NaCl, 0.05 mg ml−1 bovine serum albumin and 3.85 µM of each adapter.

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: To create a double-stranded product from the single-stranded DNA oligonucleotide, two cycles of PCR were performed using PfuUltra High-Fidelity DNA Polymerase (Agilent Technologies, Santa Clara, CA, USA) and Nextera adapter-specific primers (Supplementary Table SI2) as per the manufacturer's instructions. .. The double-stranded product was then purified using the Zymo Research DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA) and subjected to EcoRI/BamHI restriction digest using BamHI-HF (New England Biolabs) and EcoRI-HF (New England Biolabs) to prepare the construct for ligation into an EcoRI/BamHI-digested pUC19 backbone.

    Article Title: Synapse-to-Nucleus Communication through NFAT Is Mediated by L-type Ca2+ Channel Ca2+ Spike Propagation to the Soma
    Article Snippet: To generate the sGFP-NFATc4 construct, mNFATc4 ( ) was PCR amplified with primers (forward 5′-CGACTCGA GGAGGGGCCGCAAGCTGCG-3′ and reverse 5′-CGATGAATTCTCAGGCAGGAGGCTCTTCTCC-3′) to introduce a 5′ XhoI site and a 3′ EcoRI site to the resulting product, which was then gel purified. .. The mNFATc4 insert and pSGFP2-C1 backbone were digested with XhoI and EcoRI-HF (NEB) in dual restriction digests, gel purified and ligated with T4 DNA ligase (NEB).

    Article Title: Quantitative assessment of LASSO probe assembly and long-read multiplexed cloning
    Article Snippet: .. Approximately 45 μl of solution containing gel-purified fusion PCR product as described were digested by adding 5 μl of CutSmart 10X buffer (NEB) and 1 μl (20 units/μl) of EcoRI-HF (NEB) for 1 h at 37 °C followed by a denaturation step for 10 min at 80 °C. .. EcoRI digested DNA was purified with Agencourt AMPure XP beads (Beckman Coulter) and eluted in 40 μl ddH2 O.

    Article Title: The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri
    Article Snippet: Approximately 3-μ g genomic DNA was digested by EcoRI-HF (New England Biolabs, Ipswich, MA) in a 30-μ L reaction at 37°C. .. The enzyme was removed using the Wizard SV Gel and polymerase chain reaction (PCR) Clean-Up System (Promega, Madison, WI).

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: .. Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel). .. Ligation was mediated by T7 DNA ligase (NEB) to construct mUAV, Level 1 and Level 2 Acceptor Vectors.

    Article Title: Double‐digest RADseq loci using standard Illumina indexes improve deep and shallow phylogenetic resolution of Lophodermium, a widespread fungal endophyte of pine needles. Double‐digest RADseq loci using standard Illumina indexes improve deep and shallow phylogenetic resolution of Lophodermium, a widespread fungal endophyte of pine needles
    Article Snippet: Three to nine hundred nanograms of DNA per sample (6 μl total; 50–150 ng/μl) was double‐digested using the rare‐cutting EcoRI‐HF and the frequent‐cutting MseI enzymes (New England BioLabs, Ipswich, MA). .. All samples were placed randomly in PCR plates during library preparation.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: CypherSeq design and generation of empty library stocks To create the CypherSeq library construct, two 195 base PAGE Ultramer DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA) were designed (Supplementary Table SI1). .. The double-stranded product was then purified using the Zymo Research DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA) and subjected to EcoRI/BamHI restriction digest using BamHI-HF (New England Biolabs) and EcoRI-HF (New England Biolabs) to prepare the construct for ligation into an EcoRI/BamHI-digested pUC19 backbone.

    IA:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: CypherSeq design and generation of empty library stocks To create the CypherSeq library construct, two 195 base PAGE Ultramer DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA) were designed (Supplementary Table SI1). .. The double-stranded product was then purified using the Zymo Research DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA) and subjected to EcoRI/BamHI restriction digest using BamHI-HF (New England Biolabs) and EcoRI-HF (New England Biolabs) to prepare the construct for ligation into an EcoRI/BamHI-digested pUC19 backbone.

    Article Title: Calpain 5 Is Highly Expressed in the Central Nervous System (CNS), Carries Dual Nuclear Localization Signals, and Is Associated with Nuclear Promyelocytic Leukemia Protein Bodies *
    Article Snippet: Human CAPN5 cDNA (MHS1010-58128) was purchased from Thermo Scientific, Open Biosystems, Huntsville, AL. All oligonucleotides were ordered from Integrated DNA Technologies, Coralville, IA. .. EcoRI-HF and BamHI-HF were purchased from New England Biolabs, Ipswich, MA.

    Plasmid Preparation:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: The double-stranded product was then purified using the Zymo Research DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA) and subjected to EcoRI/BamHI restriction digest using BamHI-HF (New England Biolabs) and EcoRI-HF (New England Biolabs) to prepare the construct for ligation into an EcoRI/BamHI-digested pUC19 backbone. .. Digested vector and construct were run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA), and the appropriate bands were manually excised.

    Article Title: Synapse-to-Nucleus Communication through NFAT Is Mediated by L-type Ca2+ Channel Ca2+ Spike Propagation to the Soma
    Article Snippet: Paragraph title: Plasmid DNA constructs ... The mNFATc4 insert and pSGFP2-C1 backbone were digested with XhoI and EcoRI-HF (NEB) in dual restriction digests, gel purified and ligated with T4 DNA ligase (NEB).

    Article Title: Effects of claudin-1 downregulation on the physiological processes of gallbladder cancer SGC996 cells
    Article Snippet: .. R3552L and R3101L, respectively) and T4 DNA ligase used for plasmid construction were purchased from New England BioLabs, Inc. (Ipswich, MA, USA). .. Following trypsin digestion (Sangon Biotech Co., Ltd., Shanghai, China), all cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin G and 100 µg/ml streptomycin.

    Article Title: Regulatory Protein BBD18 of the Lyme Disease Spirochete: Essential Role During Tick Acquisition?
    Article Snippet: Based on BLAST alignment of WT BBD18 (B. burgdorferi B31, GenBank accession no. AE000793.2 ) with amino acid residues contacting DNA in SopB ( ) and conserved residues in other Borrelia species (B. recurrentis A1, plasmid plate 6, GenBank accession no. CP001000.1 ; B. hermsii MTW, plasmid contig 0014, GenBank accession no. CP005691.1 ), we designed four bbd18 variants, each with multiple amino acid substitutions ( ). .. These synthetic genes were excised from pUC57 with EcoRI-HF (New England Biolabs, Ipswich, MA) and ligated into appropriately digested and dephosphorylated pBSV2*flaBp ( ) in a manner following the construction of pBSV2*flaBp-bbd18, which contains the wild-type bbd18 gene under expression of the constitutive flaB promoter ( ).

    Article Title: The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri
    Article Snippet: Recipients of the reporter plasmid were selected by plating the mating mixture onto LBS with 2.5 μ g mL−1 chloramphenicol. .. Approximately 3-μ g genomic DNA was digested by EcoRI-HF (New England Biolabs, Ipswich, MA) in a 30-μ L reaction at 37°C.

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: Paragraph title: Part and vector generation ... Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel).

    Article Title: In silico analysis of the fucosylation-associated genome of the human blood fluke Schistosoma mansoni: cloning and characterization of the enzymes involved in GDP-L-fucose synthesis and Golgi import
    Article Snippet: .. The protocol for single digests of restriction-tagged GMER and the stock pGEX-6P-1 vector excluded EcoRI-HF™. .. In both schemes, reactions were incubated at 37°C for 2 h. To prevent self-ligation, 2 U calf intestinal alkaline phosphatase (CIP, New England BioLabs) in 1× NEBuffer 4 was added to the linearized pGEX-6P-1 vector, and reactions (30 μL total volume) were incubated at 37°C for 1 h. Both CIP-treated pGEX-6P-1 vector and restriction-digested GMD /GMER amplicon were purified by electrophoretic fractionation in 1% agarose gel, and the DNA fragments were isolated by QIAquick gel extraction.

    Negative Control:

    Article Title: Effects of claudin-1 downregulation on the physiological processes of gallbladder cancer SGC996 cells
    Article Snippet: The negative-control cells were transfected with the virus CON077 synthesized by Shanghai GeneChem Co., Ltd. .. R3552L and R3101L, respectively) and T4 DNA ligase used for plasmid construction were purchased from New England BioLabs, Inc. (Ipswich, MA, USA).

    Agarose Gel Electrophoresis:

    Article Title: Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador, et al. Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador
    Article Snippet: Genomic DNA of each sample (1 μg) was digested using 1 μl of EcoRI‐HF (20,000 U/ml) and 1 μl of SphI‐HF (20,000 U/ml) (New England Biolabs, Ipswitch, MA, USA) following the manufacturer's protocol. .. Barcoded samples were pooled and size‐selected between 250 and 350 bp (326–426 bp accounting for the 76 bp adapter) using a Pippin Prep 2% agarose gel cassette (Sage Science, Beverly, MA, USA).

    Spectrophotometry:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: The double-stranded product was then purified using the Zymo Research DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA) and subjected to EcoRI/BamHI restriction digest using BamHI-HF (New England Biolabs) and EcoRI-HF (New England Biolabs) to prepare the construct for ligation into an EcoRI/BamHI-digested pUC19 backbone. .. The DNA in the gel fragments was then purified using a Zymoclean Gel DNA Recovery kit (Zymo Research) and DNA was quantified using a spectrophotometer (Nanophotometer, Implen, Inc., Munich, Germany).

    Sampling:

    Article Title: Beyond the Coral Triangle: high genetic diversity and near panmixia in Singapore's populations of the broadcast spawning sea star Protoreaster nodosus
    Article Snippet: In total, 36 samples were selected from across the three main sampling localities, Pulau Sekudu, Pulau Semakau and Cyrene reefs, based on DNA quality. .. A total of 100 ng DNA from each sample was simultaneously double-digested with restriction enzymes and ligated to adapters in duplicate 13 µl reactions at 37°C for 3.5 h. Each reaction contained 5 U EcoRI-HF® (NEB), 1 U MspI, 80 U T4 DNA ligase, 1× T4 DNA ligase buffer, 50 mM NaCl, 0.05 mg ml−1 bovine serum albumin and 3.85 µM of each adapter.

    Article Title: Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador, et al. Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in Ecuador
    Article Snippet: Samples include three specimens randomly drawn within each sampling site from the monomorphic populations (with two sampling sites for Durango and San Antonio populations) and all the specimens from the polymorphic region in Otokiki (see Figure ). .. Genomic DNA of each sample (1 μg) was digested using 1 μl of EcoRI‐HF (20,000 U/ml) and 1 μl of SphI‐HF (20,000 U/ml) (New England Biolabs, Ipswitch, MA, USA) following the manufacturer's protocol.

    DNA Purification:

    Article Title: The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri
    Article Snippet: To determine the transposon insertion site within each mutant, genomic DNA was extracted from 0.5 mL overnight LBS cultures using the MasterPure DNA Purification Kit (Epicentre Biotechnologies). .. Approximately 3-μ g genomic DNA was digested by EcoRI-HF (New England Biolabs, Ipswich, MA) in a 30-μ L reaction at 37°C.

    Variant Assay:

    Article Title: Regulatory Protein BBD18 of the Lyme Disease Spirochete: Essential Role During Tick Acquisition?
    Article Snippet: Variant bbd18 genes were synthesized by GenScript Corporation (Piscataway, NJ) with EcoRI recognition sites at both ends. .. These synthetic genes were excised from pUC57 with EcoRI-HF (New England Biolabs, Ipswich, MA) and ligated into appropriately digested and dephosphorylated pBSV2*flaBp ( ) in a manner following the construction of pBSV2*flaBp-bbd18, which contains the wild-type bbd18 gene under expression of the constitutive flaB promoter ( ).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    New England Biolabs ecori hf
    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with <t>PstI</t> and <t>EcoRI</t> resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Ecori Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecori hf/product/New England Biolabs
    Average 95 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    ecori hf - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    Image Search Results


    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Journal: PLoS ONE

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    doi: 10.1371/journal.pone.0189892

    Figure Lengend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Article Snippet: Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel).

    Techniques: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    PAS136 mapping and pharynx markers. (A) Probable location of the PAS136 pharynx phenotype allele is between 6 cM and 8 cM on LG.I relative to the genetic center of the chromosome (green circle) derived by mapping with DraI or EcoRI specific SNPs corresponding to DNA clones D1007, K02B12, B0205, and F58D5 (orange lines) and between 4.64 cM and 9.2 cM (red circle) using complementation with the deficiency strains MT2179, DC1079, KR2838 and SL536 with overlapping chromosomal deletions (blue lines). (B) pha-4 RNAi used a positive control for pharynx phenotypes, arrow shows lack of myo-2::GFP in most of the head. (C) lam-3 (T22A3.8) RNAi showing a phenotype similar to PAS136 with non-adherent cells (arrow). (D) blmp-1 (F25D7.3) RNAi has a less severe PAS136 phenotype (arrow denotes cell disconnected from the pharynx). (E) hmr-1 (W02B9.1) RNAi results in a Pun phenotype with diminished anterior pharynx cells (arrow). (F) Wild-type MH27 AJM-1 adherens junction antibody staining showing pharynx (ph) and intestine (it) localization. (G) PAS136 embryo with weak and disconnect AJM-1 staining in the pharynx (ph) and more normal AJM-1 in the intestine (it). (H) Wild-type Intermediate Filaments showing three sets of marginal cells (arrows). (I) PAS136 embryo with three sets of marginal cells (arrows). Bar is ∼10 µM.

    Journal: PLoS ONE

    Article Title: Multiple Phenotypes Resulting from a Mutagenesis Screen for Pharynx Muscle Mutations in Caenorhabditis elegans

    doi: 10.1371/journal.pone.0026594

    Figure Lengend Snippet: PAS136 mapping and pharynx markers. (A) Probable location of the PAS136 pharynx phenotype allele is between 6 cM and 8 cM on LG.I relative to the genetic center of the chromosome (green circle) derived by mapping with DraI or EcoRI specific SNPs corresponding to DNA clones D1007, K02B12, B0205, and F58D5 (orange lines) and between 4.64 cM and 9.2 cM (red circle) using complementation with the deficiency strains MT2179, DC1079, KR2838 and SL536 with overlapping chromosomal deletions (blue lines). (B) pha-4 RNAi used a positive control for pharynx phenotypes, arrow shows lack of myo-2::GFP in most of the head. (C) lam-3 (T22A3.8) RNAi showing a phenotype similar to PAS136 with non-adherent cells (arrow). (D) blmp-1 (F25D7.3) RNAi has a less severe PAS136 phenotype (arrow denotes cell disconnected from the pharynx). (E) hmr-1 (W02B9.1) RNAi results in a Pun phenotype with diminished anterior pharynx cells (arrow). (F) Wild-type MH27 AJM-1 adherens junction antibody staining showing pharynx (ph) and intestine (it) localization. (G) PAS136 embryo with weak and disconnect AJM-1 staining in the pharynx (ph) and more normal AJM-1 in the intestine (it). (H) Wild-type Intermediate Filaments showing three sets of marginal cells (arrows). (I) PAS136 embryo with three sets of marginal cells (arrows). Bar is ∼10 µM.

    Article Snippet: All regions were cut with DraI (NEB) to identify SNPs, except for 2.8 LG.I the DNA was digested EcoRI-HF (NEB).

    Techniques: Derivative Assay, Clone Assay, Positive Control, Laser Capture Microdissection, Staining

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Journal: PLoS ONE

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    doi: 10.1371/journal.pone.0189892

    Figure Lengend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Article Snippet: Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel).

    Techniques: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    3C2D-qPCR design for characterizing centromere coupling. (A) Design of two primers (arrow) and one Taqman probe (ball-and-stick) to quantify the interaction between restriction fragments ligated together, each encompassing a non-homologous centromere (oval). (B) Distribution of restriction enzyme sites on fragments encompassing the centromere ( CEN ) on all 16 chromosomes using an EcoRI single digestion (3C) (left) or an EcoRI-MfeI double digestion (3C2D) (right). For each chromosome (on y-axis), the distances of the restriction sites delimitating the CEN fragment are given in kilobases (kb), in relation to the center of the CEN (x-axis). Blue vertical lines indicate EcoRI sites and red lines indicate MfeI sites.

    Journal: PLoS Genetics

    Article Title: Multiple Pairwise Analysis of Non-homologous Centromere Coupling Reveals Preferential Chromosome Size-Dependent Interactions and a Role for Bouquet Formation in Establishing the Interaction Pattern

    doi: 10.1371/journal.pgen.1006347

    Figure Lengend Snippet: 3C2D-qPCR design for characterizing centromere coupling. (A) Design of two primers (arrow) and one Taqman probe (ball-and-stick) to quantify the interaction between restriction fragments ligated together, each encompassing a non-homologous centromere (oval). (B) Distribution of restriction enzyme sites on fragments encompassing the centromere ( CEN ) on all 16 chromosomes using an EcoRI single digestion (3C) (left) or an EcoRI-MfeI double digestion (3C2D) (right). For each chromosome (on y-axis), the distances of the restriction sites delimitating the CEN fragment are given in kilobases (kb), in relation to the center of the CEN (x-axis). Blue vertical lines indicate EcoRI sites and red lines indicate MfeI sites.

    Article Snippet: For the double digestion, EcoRI-HF and MfeI-HF (NEB) generated cohesive ends that were compatible, yet recognizing slightly different sequences (GAATTC and CAATTG respectively).

    Techniques: Real-time Polymerase Chain Reaction