drai  (New England Biolabs)


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  • 99
    Name:
    DraI
    Description:
    DraI 10 000 units
    Catalog Number:
    R0129L
    Price:
    264
    Size:
    10 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs drai
    DraI
    DraI 10 000 units
    https://www.bioz.com/result/drai/product/New England Biolabs
    Average 99 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    drai - by Bioz Stars, 2019-08
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Sema3d controls collective endothelial cell migration by distinct mechanisms via Nrp1 and PlxnD1
    Article Snippet: Annealed CRISPR target site primers (CRIsema3d SE, 5′-TAGGAGACAGGTCAGGACGGGA-3′, and AS, 5′-AAACTCCCGTCCTGACCTGTCT-3′) were cloned into the guide RNA (gRNA) expression vector pDR274 as previously described ( ). .. After DraI digestion (New England Biolabs, Inc.), gRNA was transcribed using MEGAshortscript T7 kit (Ambion).

    Article Title: Evidence that RpoS (?S) in Borrelia burgdorferi Is Controlled Directly by RpoN (?54/?N)
    Article Snippet: The BD SMART IIA primer, which anneals to the BD SMART IIA oligonucleotide linked to the 3′ end of the first-strand cDNA, and an rpoS -specific primer upstream of the gene-specific primers used in first-strand cDNA synthesis (rpoSR125) (Table ) were used to amplify the resulting cDNAs. .. PCR-amplified inserts cloned into the pGEM-T Easy vector (Promega, Madison, WI) were digested with the restriction endonuclease DraI (New England Biolabs, Ipswich, MA) to verify that they represented rpoS . .. Seventeen of 25 clones contained rpoS -specific sequences, as determined by the DraI restriction pattern (not shown).

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: The clonal N40 parent isolate (cN40) and each of the derived isolates were grown from frozen stocks at 34°C in BSK-H medium supplemented with 6% rabbit serum (Sigma Aldrich) to a density of ∼5*107 cells/ml and the genomic DNA was purified using the DNeasy Blood and Tissue Kit protocol for gram negative bacteria (Qiagen; Valencia, CA). .. The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. Long-range PCR amplification was conducted using the primers N40-vlsLR-R (5′ Phos - GCT GGA CTT GAA TTT GGT AGG GAT TC 3′ ) and N40-vlsLR-F (5′ Phos - GGT GAT GGT GCC GAT TCA AAA TCT GG 3′ ) which anneal to the unique conserved regions flanking the unexpressed cassettes.

    Centrifugation:

    Article Title: Polymorphisms in thymidylate synthase gene and susceptibility to breast cancer in a Chinese population: a case-control analysis
    Article Snippet: By centrifugation of 5-ml whole blood, genomic DNA was extracted from the leukocyte pellet obtained from the buffy coat of each blood sample. .. For the TS 3'-UTR del6 polymorphism, 152 bp (i.e. del6) or 158 bp fragments (i.e. ins6) were amplified and then digested by Dra I (New England BioLabs, Inc., Beverly, MA).

    Amplification:

    Article Title: SMN1 dosage analysis in spinal muscular atrophy from India
    Article Snippet: Each patient sample was analyzed in duplicate, and controls included 5 known two-copy, a one-copy and a homozygous deleted DNA samples, as well as water (no DNA template control). .. After PCR, 4 μl of amplified DNA from each reaction was digested in a 6 μl reaction volume with 1 unit of Dra I (New England Biolabs, USA) for 3 hours at 37°C. .. Two μl of the Dra I digestion were mixed with 4 μl of formamide, 0.5 μl of Gene Scan-500 Rox (ABI) and 0.5 μl of loading buffer, heated at 95°C for 5 min and then placed on ice for 2 min. Digested PCR product mixtures were analyzed on an ABI 373 Genetic Analyzer instrument (ABI).

    Article Title: Disease progression in patients with single, large-scale mitochondrial DNA deletions
    Article Snippet: PCR reactions used ∼100 ng of DNA that was added to PCR mastermix [distilled H2 O, LA Taq buffer (TaKaRa), 10 mM dNTPs, 20 mM forward and reverse primers and 2.5 units of LA Taq enzyme (TaKaRa)] to a total volume of 50 μl and subjected to the following cycling conditions: 94°C for 1 min; 35 cycles of 94°C for 30 s, 58°C for 30 s and 68°C for 11 min; final extension of 72°C for 10 min. Amplified products were separated through a 0.7% agarose gel, using a 1 kb DNA ladder to estimate product size and determine mitochondrial DNA deletion sizes. .. PCR amplimers (5 µl) were digested to a series of DNA fragments of known length and position within the mitochondrial genome using restriction enzymes XhoI, BamHI, XcmI and DraI (New England Biolabs) before separation through a 0.7% agarose gel.

    Article Title: Polymorphisms in thymidylate synthase gene and susceptibility to breast cancer in a Chinese population: a case-control analysis
    Article Snippet: For the TSER polymorphism, 243 bp (i.e. 3R), 215 bp (i.e. 2R), 271 bp (i.e. 4R) and 299 bp (i.e. 5R) fragments were identified and separated on 3% agarose gels. .. For the TS 3'-UTR del6 polymorphism, 152 bp (i.e. del6) or 158 bp fragments (i.e. ins6) were amplified and then digested by Dra I (New England BioLabs, Inc., Beverly, MA). .. The variant (ins6) allele produces two fragments of 88 and 70 bp, while the wild-type (del6) allele produces a single 152-bp fragment.

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: The specific process used for amplifying circularized asMIPs was tailored to the method of quantitation; for arrays, MIPs were amplified with biotinylated PCR primers before being hybridized to microarrays. .. PCR reactions were carried out using the following three-step thermal profile: denaturation at 94°C for 15 seconds, annealing at 60°C for 1 minute and extension at 72°C for 5 s. The resulting PCR products were treated with 20 u of DraI (NEB catalog no. R0129S) incubated at 37°C for 1 hour; DraI digestion was used to separate the two labeled sequence tags, as well as remove the now extraneous interrogation sequences, which could confound hybridization.

    Article Title: Genetic Tests for Ecological and Allopatric Speciation in Anoles on an Island Archipelago
    Article Snippet: The cyt b fragment used in the phylogeographic analysis was digested after amplification using the restriction enzyme SspI (New England Biolabs) for 3 hours at 37°. .. To further distinguish between southwest and northwest lineages, we digested the same fragment using the restriction enzyme DraI (New England Biolabs) that cuts the PCR products from the northwest lineage at position 227, while those from SW lineage were uncut by this enzyme.

    Article Title: The genetics of feed conversion efficiency traits in a commercial broiler line
    Article Snippet: In brief, genomic regions were amplified using primer combinations ggaAGK_f2/ggaAGK_r2 or ggaGTF2I_f1/ggaGTF2I_r1 (see ) in a standard PCR mix with SupraTherm Taq Polymerase (Genecraft, Lüdinghausen, Germany) as described elsewhere . .. Overnight digestion of amplification products was performed using 5 U/μl Dra I and Nsi I restriction enzymes (New England Biolabs, Frankfurt, Germany) for GTF2I SNP c.2011 A > C and AGK SNP c.1166 G > A, respectively. .. Restriction fragments were separated on 2% agarose gel and analyzed.

    Article Title: Diet adaptation in dog reflects spread of prehistoric agriculture
    Article Snippet: DNA was digested with restriction enzyme DRAI (New England Biolabs, Ipswich, MA, USA) to separate individual amylase copies to allow for better partitioning, except for DNA eluted from FTA cards as the elution method (boiling) is expected to have sheared the DNA sufficiently. .. DNA was digested with restriction enzyme DRAI (New England Biolabs, Ipswich, MA, USA) to separate individual amylase copies to allow for better partitioning, except for DNA eluted from FTA cards as the elution method (boiling) is expected to have sheared the DNA sufficiently.

    Article Title: Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice
    Article Snippet: For the analysis of Spp1 exons 3 and 4, PCR amplification was carried out using the Spp1 ex3-4 F and R primers listed in Table S3 . .. The PCR products were purified and analyzed by RFLP analyses using Nar I or Dra I (New England Biolabs Japan).

    Article Title: MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4
    Article Snippet: For CD1 (NM_053056.2) and PAK4 (Variant 5, NM_001014834.2) full-length 3’ UTR luciferase reporter constructs were generated. .. PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA). .. The PAK4 constructs containing mutations at the sequence of binding region of potential binding sites were generated using a site directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA).

    Article Title: Enrichment of Human Hematopoietic Stem/Progenitor Cells Facilitates Transduction for Stem Cell Gene Therapy
    Article Snippet: Reaction mixtures were prepared consisting of 22 μl volumes containing 1× ddPCR Master Mix (Bio-Rad, Hercules, CA), primers, and probe specific to either the HIV-1 Psi region, to detect all vectors, or to each of the fluorescent reporter genes (400 nM and 100 nM for primers and probe, respectively), DraI (40 U; New England Biolabs, Ipswich, MA), and 1.1 μl (4 μl for cfu) of the genomic DNA sample. .. Reaction mixtures were prepared consisting of 22 μl volumes containing 1× ddPCR Master Mix (Bio-Rad, Hercules, CA), primers, and probe specific to either the HIV-1 Psi region, to detect all vectors, or to each of the fluorescent reporter genes (400 nM and 100 nM for primers and probe, respectively), DraI (40 U; New England Biolabs, Ipswich, MA), and 1.1 μl (4 μl for cfu) of the genomic DNA sample.

    Article Title: Mutations in monoamine oxidase (MAO) genes in mice lead to hypersensitivity to serotonin-enhancing drugs: implications for drug side effects in humans
    Article Snippet: PCR conditions were: 95 °C 15 min, (94 °C 30 s, 55 °C 30 s, 72 °C 1 min) ×35 cycles and 72 °C 5 min. .. The PCR product from the MAOA reaction was digested with Dra I (New England Biolabs, Ipswich, MA, USA) at 37 °C for 2 h. The amplified DNA product after Dra I digestion exhibits a 100- and 200-base pair (bp) product for WT mice, three bands of 100, 200 and 300 bps for heterozygous female mice and an uncut 300-bp product for MAOA mutant mice. .. For the MAOB reaction, primers were: 5′-CTACAAAGCA GATTGCCACGC-3′ and 5′-TACCTGACATCAACTGGTCCC-3′.

    Article Title: Evidence that RpoS (?S) in Borrelia burgdorferi Is Controlled Directly by RpoN (?54/?N)
    Article Snippet: Determination of the initiation of transcription for a given gene can provide strategic information for identifying a gene's nearby promoter. rpoS transcripts of B. burgdorferi BbAH130 ( ) were reverse transcribed in BD SMART-RACE (switching mechanism at 5′ end of RNA transcript-rapid amplification of cDNA ends) reactions (BD Biosciences, San Jose, CA) using two rpoS -specific primers (rpoSR422 and rpoSR232) (Table ), which are 422 and 232 bases, respectively, downstream of the rpoS translation start site. .. PCR-amplified inserts cloned into the pGEM-T Easy vector (Promega, Madison, WI) were digested with the restriction endonuclease DraI (New England Biolabs, Ipswich, MA) to verify that they represented rpoS .

    Article Title: Parallel gene analysis with allele-specific padlock probes and tag microarrays
    Article Snippet: Paragraph title: Ligation, exonuclease treatment and amplification ... Genomic DNA was reduced in size by digestion using DraI and SspI (New England Biolabs) for 2 h in the recommended buffers.

    Article Title: Differential microRNA epression in asthma and the role of miR-1248 in regulation of IL-5
    Article Snippet: To clone IL-5 3’UTR into the pmirGLO vector (Promega), the IL-5 3’UTR was amplified from total cDNA from PBMCs using the following primers: IL-5 3’UTR DraI forward: 5’-AAA TTT AAA AGA CTA AAC TGG TTT GTT GCA GC-3’; IL-5 3’UTR SalI reverse: 5’-AAA GTC GAC GAA CAG TTG TCT ATT TTT GTT TTA TTA GA-3’. .. The vector and PCR product were digested with DraI and SalI (both from New England Biolabs), gel purified, and ligated with Quick T4 DNA Ligase (New England Biolabs).

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. Long-range PCR amplification was conducted using the primers N40-vlsLR-R (5′ Phos - GCT GGA CTT GAA TTT GGT AGG GAT TC 3′ ) and N40-vlsLR-F (5′ Phos - GGT GAT GGT GCC GAT TCA AAA TCT GG 3′ ) which anneal to the unique conserved regions flanking the unexpressed cassettes.

    Reporter Assay:

    Article Title: MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4
    Article Snippet: Paragraph title: Luciferase reporter assay ... PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA).

    Positive Control:

    Article Title: Polymorphisms in thymidylate synthase gene and susceptibility to breast cancer in a Chinese population: a case-control analysis
    Article Snippet: For the TS 3'-UTR del6 polymorphism, 152 bp (i.e. del6) or 158 bp fragments (i.e. ins6) were amplified and then digested by Dra I (New England BioLabs, Inc., Beverly, MA). .. The variant (ins6) allele produces two fragments of 88 and 70 bp, while the wild-type (del6) allele produces a single 152-bp fragment.

    Salting Out:

    Article Title: Association between two common polymorphisms (single nucleotide polymorphism -250G/A and -514C/T) of the hepatic lipase gene and coronary artery disease in type 2 diabetic patients
    Article Snippet: DNA was extracted from leukocytes, taken from peripheral blood, by using a salting-out procedure (Miller's method). .. [ ] Genotyping of -250G/A (rs2070895), and -514C/T (rs1800588) SNPs, in the promoter region of the LIPC gene were performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism, followed by digestion with the restriction enzymes DraI and NlaIII (New England Biolabs), respectively.

    Autoradiography:

    Article Title: Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells
    Article Snippet: 2.4 Total DNA was digested with DraI (New England Biolabs), and then precipitated and suspended in 10 mM Hepes-NaOH (pH 7.2). .. 2.4 Total DNA was digested with DraI (New England Biolabs), and then precipitated and suspended in 10 mM Hepes-NaOH (pH 7.2).

    GWAS:

    Article Title: The genetics of feed conversion efficiency traits in a commercial broiler line
    Article Snippet: Overnight digestion of amplification products was performed using 5 U/μl Dra I and Nsi I restriction enzymes (New England Biolabs, Frankfurt, Germany) for GTF2I SNP c.2011 A > C and AGK SNP c.1166 G > A, respectively. .. Overnight digestion of amplification products was performed using 5 U/μl Dra I and Nsi I restriction enzymes (New England Biolabs, Frankfurt, Germany) for GTF2I SNP c.2011 A > C and AGK SNP c.1166 G > A, respectively.

    Construct:

    Article Title: MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4
    Article Snippet: For CD1 (NM_053056.2) and PAK4 (Variant 5, NM_001014834.2) full-length 3’ UTR luciferase reporter constructs were generated. .. PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA).

    Real-time Polymerase Chain Reaction:

    Article Title: Diet adaptation in dog reflects spread of prehistoric agriculture
    Article Snippet: Compared with real-time PCR, ddPCR has been shown to reduce mean coefficients of variation by 37–86% and improve reproducibility by a factor of seven ( ). .. DNA was digested with restriction enzyme DRAI (New England Biolabs, Ipswich, MA, USA) to separate individual amylase copies to allow for better partitioning, except for DNA eluted from FTA cards as the elution method (boiling) is expected to have sheared the DNA sufficiently.

    Microarray:

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: Paragraph title: Microarray sample preparation ... PCR reactions were carried out using the following three-step thermal profile: denaturation at 94°C for 15 seconds, annealing at 60°C for 1 minute and extension at 72°C for 5 s. The resulting PCR products were treated with 20 u of DraI (NEB catalog no. R0129S) incubated at 37°C for 1 hour; DraI digestion was used to separate the two labeled sequence tags, as well as remove the now extraneous interrogation sequences, which could confound hybridization.

    Incubation:

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: Specifically, 1 μl of eluted asMIPs were added to a 50 μl PCR reaction containing 5'-biotinylated P1 and P2 (P1: bio-CGAACGACGA CATAGACCAC, P2: bio-CGCTTTAGGT GCAGACACAA) primers at 0.6 μM concentration and 1× Platinum PCR SuperMix (Invitrogen, catalog no. 11306-016). .. PCR reactions were carried out using the following three-step thermal profile: denaturation at 94°C for 15 seconds, annealing at 60°C for 1 minute and extension at 72°C for 5 s. The resulting PCR products were treated with 20 u of DraI (NEB catalog no. R0129S) incubated at 37°C for 1 hour; DraI digestion was used to separate the two labeled sequence tags, as well as remove the now extraneous interrogation sequences, which could confound hybridization. .. Unique primers were used to amplify asMIPs quantified using Illumina HTS; in all other respects the PCR reaction conditions were identical to those specified for microarray sample preparation.

    Article Title: Enrichment of Human Hematopoietic Stem/Progenitor Cells Facilitates Transduction for Stem Cell Gene Therapy
    Article Snippet: After antibody incubation, RBCs were lysed using BD FACS-Lysing Solution (BD Biosciences). .. Reaction mixtures were prepared consisting of 22 μl volumes containing 1× ddPCR Master Mix (Bio-Rad, Hercules, CA), primers, and probe specific to either the HIV-1 Psi region, to detect all vectors, or to each of the fluorescent reporter genes (400 nM and 100 nM for primers and probe, respectively), DraI (40 U; New England Biolabs, Ipswich, MA), and 1.1 μl (4 μl for cfu) of the genomic DNA sample.

    Article Title: Parallel gene analysis with allele-specific padlock probes and tag microarrays
    Article Snippet: Genomic DNA was reduced in size by digestion using DraI and SspI (New England Biolabs) for 2 h in the recommended buffers. .. After ligation, 10 µl of exonuclease mix was added to the reactions for a final concentration of 67 mM Tris–HCl pH 9.0, 50 mM KCl, 6.7 mM MgCl2 , 0.05 µg/µl BSA, 0.35 U/µl exonuclease I (New England Biolabs) and 0.35 U/µl exonuclease III (Amersham Biosciences).

    Article Title: Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1
    Article Snippet: Briefly, the heteroduplex phagemid DNA substrate (48 fmol) containing a T/G mismatch in its unique SalI site and a single nick generated by Nt·BstNBI 361 nucleotides 5′ from the mispaired T was incubated with 100 μg of nuclear extracts of HEK293 cells pretreated or not with EXO1 siRNA and supplemented with 40 nM Exo1 wt, E109K or D173A in 20 mM Tris·HCl pH 7.6, 110 mM KCl, 5 mM MgCl2 , 1 mM glutathione, 1.5 mM ATP, 50 μg/ml BSA and 100 μM dNTPs for 30 min in a total volume of 25 μl. .. The reactions were terminated by a 30-min incubation with a stop solution (final concentrations: 0.5 mM EDTA, 1.5% SDS (sodium dodecyl sulfate), 2.5 mg/ml proteinase K), cleaned up on a MinElute column (Qiagen), and the recovered phagemid was subjected to restriction digest with 6 U SalI and 20 U DraI (NEB). .. RNase A (40 ng, Sigma-Aldrich) was then added and, following an overnight incubation at 37°C, the reaction products were separated on a 1% agarose gel eluted with TAE buffer and stained with GelRed.

    Luciferase:

    Article Title: MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4
    Article Snippet: Paragraph title: Luciferase reporter assay ... PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA).

    Activity Assay:

    Article Title: MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4
    Article Snippet: PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA). .. PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA).

    Expressing:

    Article Title: Sema3d controls collective endothelial cell migration by distinct mechanisms via Nrp1 and PlxnD1
    Article Snippet: Annealed CRISPR target site primers (CRIsema3d SE, 5′-TAGGAGACAGGTCAGGACGGGA-3′, and AS, 5′-AAACTCCCGTCCTGACCTGTCT-3′) were cloned into the guide RNA (gRNA) expression vector pDR274 as previously described ( ). .. After DraI digestion (New England Biolabs, Inc.), gRNA was transcribed using MEGAshortscript T7 kit (Ambion).

    Article Title: MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4
    Article Snippet: For CD1 (NM_053056.2) and PAK4 (Variant 5, NM_001014834.2) full-length 3’ UTR luciferase reporter constructs were generated. .. PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA). .. The PAK4 constructs containing mutations at the sequence of binding region of potential binding sites were generated using a site directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA).

    Article Title: Enrichment of Human Hematopoietic Stem/Progenitor Cells Facilitates Transduction for Stem Cell Gene Therapy
    Article Snippet: From among the huCD45+ cells, expression of mCitrine, mStrawberry, and mCerulean was analyzed using flow cytometry. .. Reaction mixtures were prepared consisting of 22 μl volumes containing 1× ddPCR Master Mix (Bio-Rad, Hercules, CA), primers, and probe specific to either the HIV-1 Psi region, to detect all vectors, or to each of the fluorescent reporter genes (400 nM and 100 nM for primers and probe, respectively), DraI (40 U; New England Biolabs, Ipswich, MA), and 1.1 μl (4 μl for cfu) of the genomic DNA sample.

    Transplantation Assay:

    Article Title: Enrichment of Human Hematopoietic Stem/Progenitor Cells Facilitates Transduction for Stem Cell Gene Therapy
    Article Snippet: Paragraph title: Transplantation of Transduced Human CB CD34+ /CD38− Cells in Immune-Deficient Mice ... Reaction mixtures were prepared consisting of 22 μl volumes containing 1× ddPCR Master Mix (Bio-Rad, Hercules, CA), primers, and probe specific to either the HIV-1 Psi region, to detect all vectors, or to each of the fluorescent reporter genes (400 nM and 100 nM for primers and probe, respectively), DraI (40 U; New England Biolabs, Ipswich, MA), and 1.1 μl (4 μl for cfu) of the genomic DNA sample.

    Transformation Assay:

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. BigEasy vectors containing an intact cassette region were amplified in E.coli and isolated using a Qiagen Mini-prep kit.

    Derivative Assay:

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: The clonal N40 parent isolate (cN40) and each of the derived isolates were grown from frozen stocks at 34°C in BSK-H medium supplemented with 6% rabbit serum (Sigma Aldrich) to a density of ∼5*107 cells/ml and the genomic DNA was purified using the DNeasy Blood and Tissue Kit protocol for gram negative bacteria (Qiagen; Valencia, CA). .. The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. Long-range PCR amplification was conducted using the primers N40-vlsLR-R (5′ Phos - GCT GGA CTT GAA TTT GGT AGG GAT TC 3′ ) and N40-vlsLR-F (5′ Phos - GGT GAT GGT GCC GAT TCA AAA TCT GG 3′ ) which anneal to the unique conserved regions flanking the unexpressed cassettes.

    Hybridization:

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: Specifically, 1 μl of eluted asMIPs were added to a 50 μl PCR reaction containing 5'-biotinylated P1 and P2 (P1: bio-CGAACGACGA CATAGACCAC, P2: bio-CGCTTTAGGT GCAGACACAA) primers at 0.6 μM concentration and 1× Platinum PCR SuperMix (Invitrogen, catalog no. 11306-016). .. PCR reactions were carried out using the following three-step thermal profile: denaturation at 94°C for 15 seconds, annealing at 60°C for 1 minute and extension at 72°C for 5 s. The resulting PCR products were treated with 20 u of DraI (NEB catalog no. R0129S) incubated at 37°C for 1 hour; DraI digestion was used to separate the two labeled sequence tags, as well as remove the now extraneous interrogation sequences, which could confound hybridization. .. Unique primers were used to amplify asMIPs quantified using Illumina HTS; in all other respects the PCR reaction conditions were identical to those specified for microarray sample preparation.

    Article Title: Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells
    Article Snippet: Paragraph title: DNA modification, 2D-AGE and Southern hybridisation ... 2.4 Total DNA was digested with DraI (New England Biolabs), and then precipitated and suspended in 10 mM Hepes-NaOH (pH 7.2).

    Flow Cytometry:

    Article Title: Enrichment of Human Hematopoietic Stem/Progenitor Cells Facilitates Transduction for Stem Cell Gene Therapy
    Article Snippet: From among the huCD45+ cells, expression of mCitrine, mStrawberry, and mCerulean was analyzed using flow cytometry. .. Reaction mixtures were prepared consisting of 22 μl volumes containing 1× ddPCR Master Mix (Bio-Rad, Hercules, CA), primers, and probe specific to either the HIV-1 Psi region, to detect all vectors, or to each of the fluorescent reporter genes (400 nM and 100 nM for primers and probe, respectively), DraI (40 U; New England Biolabs, Ipswich, MA), and 1.1 μl (4 μl for cfu) of the genomic DNA sample.

    Gas Chromatography:

    Article Title: Evidence that RpoS (?S) in Borrelia burgdorferi Is Controlled Directly by RpoN (?54/?N)
    Article Snippet: PCR-amplified inserts cloned into the pGEM-T Easy vector (Promega, Madison, WI) were digested with the restriction endonuclease DraI (New England Biolabs, Ipswich, MA) to verify that they represented rpoS . .. For five of these eight clones, residue −50 upstream of the rpoS ORF represented the transcript start site by RACE analysis, whereas there was no consensus residue for the other three sequences.

    Article Title: Differential microRNA epression in asthma and the role of miR-1248 in regulation of IL-5
    Article Snippet: To clone IL-5 3’UTR into the pmirGLO vector (Promega), the IL-5 3’UTR was amplified from total cDNA from PBMCs using the following primers: IL-5 3’UTR DraI forward: 5’-AAA TTT AAA AGA CTA AAC TGG TTT GTT GCA GC-3’; IL-5 3’UTR SalI reverse: 5’-AAA GTC GAC GAA CAG TTG TCT ATT TTT GTT TTA TTA GA-3’. .. The vector and PCR product were digested with DraI and SalI (both from New England Biolabs), gel purified, and ligated with Quick T4 DNA Ligase (New England Biolabs).

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. Long-range PCR amplification was conducted using the primers N40-vlsLR-R (5′ Phos - GCT GGA CTT GAA TTT GGT AGG GAT TC 3′ ) and N40-vlsLR-F (5′ Phos - GGT GAT GGT GCC GAT TCA AAA TCT GG 3′ ) which anneal to the unique conserved regions flanking the unexpressed cassettes.

    Ligation:

    Article Title: Parallel gene analysis with allele-specific padlock probes and tag microarrays
    Article Snippet: Paragraph title: Ligation, exonuclease treatment and amplification ... Genomic DNA was reduced in size by digestion using DraI and SspI (New England Biolabs) for 2 h in the recommended buffers.

    Digital PCR:

    Article Title: Diet adaptation in dog reflects spread of prehistoric agriculture
    Article Snippet: We used droplet digital PCR (ddPCR) to quantify individual AMY2B copy numbers in all dogs except the Dingoes (see below). .. DNA was digested with restriction enzyme DRAI (New England Biolabs, Ipswich, MA, USA) to separate individual amylase copies to allow for better partitioning, except for DNA eluted from FTA cards as the elution method (boiling) is expected to have sheared the DNA sufficiently.

    Generated:

    Article Title: MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4
    Article Snippet: For CD1 (NM_053056.2) and PAK4 (Variant 5, NM_001014834.2) full-length 3’ UTR luciferase reporter constructs were generated. .. PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA).

    Article Title: Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1
    Article Snippet: Briefly, the heteroduplex phagemid DNA substrate (48 fmol) containing a T/G mismatch in its unique SalI site and a single nick generated by Nt·BstNBI 361 nucleotides 5′ from the mispaired T was incubated with 100 μg of nuclear extracts of HEK293 cells pretreated or not with EXO1 siRNA and supplemented with 40 nM Exo1 wt, E109K or D173A in 20 mM Tris·HCl pH 7.6, 110 mM KCl, 5 mM MgCl2 , 1 mM glutathione, 1.5 mM ATP, 50 μg/ml BSA and 100 μM dNTPs for 30 min in a total volume of 25 μl. .. The reactions were terminated by a 30-min incubation with a stop solution (final concentrations: 0.5 mM EDTA, 1.5% SDS (sodium dodecyl sulfate), 2.5 mg/ml proteinase K), cleaned up on a MinElute column (Qiagen), and the recovered phagemid was subjected to restriction digest with 6 U SalI and 20 U DraI (NEB).

    DNA Sequencing:

    Article Title: Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice
    Article Snippet: The PCR products were purified and analyzed by RFLP analyses using Nar I or Dra I (New England Biolabs Japan). .. The PCR products were purified and analyzed by RFLP analyses using Nar I or Dra I (New England Biolabs Japan).

    Article Title: MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4
    Article Snippet: PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA). .. PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA).

    Sequencing:

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: Specifically, 1 μl of eluted asMIPs were added to a 50 μl PCR reaction containing 5'-biotinylated P1 and P2 (P1: bio-CGAACGACGA CATAGACCAC, P2: bio-CGCTTTAGGT GCAGACACAA) primers at 0.6 μM concentration and 1× Platinum PCR SuperMix (Invitrogen, catalog no. 11306-016). .. PCR reactions were carried out using the following three-step thermal profile: denaturation at 94°C for 15 seconds, annealing at 60°C for 1 minute and extension at 72°C for 5 s. The resulting PCR products were treated with 20 u of DraI (NEB catalog no. R0129S) incubated at 37°C for 1 hour; DraI digestion was used to separate the two labeled sequence tags, as well as remove the now extraneous interrogation sequences, which could confound hybridization. .. Unique primers were used to amplify asMIPs quantified using Illumina HTS; in all other respects the PCR reaction conditions were identical to those specified for microarray sample preparation.

    Article Title: Genetic Tests for Ecological and Allopatric Speciation in Anoles on an Island Archipelago
    Article Snippet: The lineages were first investigated using complete cytochrome b sequence from the mtDNA. .. To further distinguish between southwest and northwest lineages, we digested the same fragment using the restriction enzyme DraI (New England Biolabs) that cuts the PCR products from the northwest lineage at position 227, while those from SW lineage were uncut by this enzyme.

    Article Title: Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice
    Article Snippet: The PCR products were purified and analyzed by RFLP analyses using Hpy CH4III, and then direct sequencing was performed as described above. .. The PCR products were purified and analyzed by RFLP analyses using Nar I or Dra I (New England Biolabs Japan).

    Article Title: MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4
    Article Snippet: PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA). .. PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA).

    Article Title: Differential microRNA epression in asthma and the role of miR-1248 in regulation of IL-5
    Article Snippet: The vector and PCR product were digested with DraI and SalI (both from New England Biolabs), gel purified, and ligated with Quick T4 DNA Ligase (New England Biolabs). .. Following transformation into NEB-5-α cells, samples were plated onto an ampicillin-containing agar plate, and colonies were selected and grown in 5 ml of Luria-Bertani broth.

    Injection:

    Article Title: Sema3d controls collective endothelial cell migration by distinct mechanisms via Nrp1 and PlxnD1
    Article Snippet: After DraI digestion (New England Biolabs, Inc.), gRNA was transcribed using MEGAshortscript T7 kit (Ambion). .. Nls-zCas9-nls mRNA was synthesized as previously described ( ).

    Recombinant:

    Article Title: HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner
    Article Snippet: Recombinant HIV-1 wild-type NCp(1-55) (i.e. NCp7) and NCp15 (based on sequences from GenBank, accession number AF324493) were expressed and purified as described – . .. AlwNI , DraI and Mo-MuLV RT enzymes were purchased from New England Biolabs (Ipswich, MA).

    Staining:

    Article Title: Association between two common polymorphisms (single nucleotide polymorphism -250G/A and -514C/T) of the hepatic lipase gene and coronary artery disease in type 2 diabetic patients
    Article Snippet: [ ] Genotyping of -250G/A (rs2070895), and -514C/T (rs1800588) SNPs, in the promoter region of the LIPC gene were performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism, followed by digestion with the restriction enzymes DraI and NlaIII (New England Biolabs), respectively. .. Thermal cycling conditions were similar for two SNPs and were as follows: An initial denaturation step at 94°C for 5 min, 35 cycles at 94°C for 30 s, 59 or 57°C for 30 s (depend on primers), 72°C for 45 s and a final extension step at 72°C for 5 min.

    Mutagenesis:

    Article Title: Sema3d controls collective endothelial cell migration by distinct mechanisms via Nrp1 and PlxnD1
    Article Snippet: Paragraph title: Generation of sema3d mutants by CRISPR/Cas9-mediated mutagenesis ... After DraI digestion (New England Biolabs, Inc.), gRNA was transcribed using MEGAshortscript T7 kit (Ambion).

    Article Title: Mutations in monoamine oxidase (MAO) genes in mice lead to hypersensitivity to serotonin-enhancing drugs: implications for drug side effects in humans
    Article Snippet: PCR conditions were: 95 °C 15 min, (94 °C 30 s, 55 °C 30 s, 72 °C 1 min) ×35 cycles and 72 °C 5 min. .. The PCR product from the MAOA reaction was digested with Dra I (New England Biolabs, Ipswich, MA, USA) at 37 °C for 2 h. The amplified DNA product after Dra I digestion exhibits a 100- and 200-base pair (bp) product for WT mice, three bands of 100, 200 and 300 bps for heterozygous female mice and an uncut 300-bp product for MAOA mutant mice. .. For the MAOB reaction, primers were: 5′-CTACAAAGCA GATTGCCACGC-3′ and 5′-TACCTGACATCAACTGGTCCC-3′.

    Isolation:

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. The PCR reactions contained 2–6 ng/µl of genomic DNA, 0.2 mM of each dNTP, 0.4 µM of each primer, 7% DMSO, 1× GC buffer and 0.02 U/µl of Phusion Hot Start II DNA Polymerase (NEB) and were amplified with 25 cycles of 30 s at 98°C and 90 s at 72°C.

    Size-exclusion Chromatography:

    Article Title: SMN1 dosage analysis in spinal muscular atrophy from India
    Article Snippet: PCR amplification conditions were as follows: initial denaturation at 95°C for 5 min followed by 22 cycles of denaturation for 30 sec at 95°C, annealing for 30 s at 55°C, and elongation for 2 min at 72°C, followed by a final extension step for 10 min at 72°C. .. After PCR, 4 μl of amplified DNA from each reaction was digested in a 6 μl reaction volume with 1 unit of Dra I (New England Biolabs, USA) for 3 hours at 37°C.

    Labeling:

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: Specifically, 1 μl of eluted asMIPs were added to a 50 μl PCR reaction containing 5'-biotinylated P1 and P2 (P1: bio-CGAACGACGA CATAGACCAC, P2: bio-CGCTTTAGGT GCAGACACAA) primers at 0.6 μM concentration and 1× Platinum PCR SuperMix (Invitrogen, catalog no. 11306-016). .. PCR reactions were carried out using the following three-step thermal profile: denaturation at 94°C for 15 seconds, annealing at 60°C for 1 minute and extension at 72°C for 5 s. The resulting PCR products were treated with 20 u of DraI (NEB catalog no. R0129S) incubated at 37°C for 1 hour; DraI digestion was used to separate the two labeled sequence tags, as well as remove the now extraneous interrogation sequences, which could confound hybridization. .. Unique primers were used to amplify asMIPs quantified using Illumina HTS; in all other respects the PCR reaction conditions were identical to those specified for microarray sample preparation.

    Purification:

    Article Title: HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner
    Article Snippet: Mo-MuLV NCp10 was expressed and purified essentially as described above for HIV-1 NCp7. .. AlwNI , DraI and Mo-MuLV RT enzymes were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice
    Article Snippet: Each PCR product was subjected to automatic electrophoresis using MultiNA. .. The PCR products were purified and analyzed by RFLP analyses using Nar I or Dra I (New England Biolabs Japan). .. The PCR products identified as positive were analyzed by direct sequencing.

    Article Title: Differential microRNA epression in asthma and the role of miR-1248 in regulation of IL-5
    Article Snippet: To clone IL-5 3’UTR into the pmirGLO vector (Promega), the IL-5 3’UTR was amplified from total cDNA from PBMCs using the following primers: IL-5 3’UTR DraI forward: 5’-AAA TTT AAA AGA CTA AAC TGG TTT GTT GCA GC-3’; IL-5 3’UTR SalI reverse: 5’-AAA GTC GAC GAA CAG TTG TCT ATT TTT GTT TTA TTA GA-3’. .. The vector and PCR product were digested with DraI and SalI (both from New England Biolabs), gel purified, and ligated with Quick T4 DNA Ligase (New England Biolabs). .. Following transformation into NEB-5-α cells, samples were plated onto an ampicillin-containing agar plate, and colonies were selected and grown in 5 ml of Luria-Bertani broth.

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: The clonal N40 parent isolate (cN40) and each of the derived isolates were grown from frozen stocks at 34°C in BSK-H medium supplemented with 6% rabbit serum (Sigma Aldrich) to a density of ∼5*107 cells/ml and the genomic DNA was purified using the DNeasy Blood and Tissue Kit protocol for gram negative bacteria (Qiagen; Valencia, CA). .. The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B).

    Cytometry:

    Article Title: Enrichment of Human Hematopoietic Stem/Progenitor Cells Facilitates Transduction for Stem Cell Gene Therapy
    Article Snippet: From among the huCD45+ cells, expression of mCitrine, mStrawberry, and mCerulean was analyzed using flow cytometry. .. Reaction mixtures were prepared consisting of 22 μl volumes containing 1× ddPCR Master Mix (Bio-Rad, Hercules, CA), primers, and probe specific to either the HIV-1 Psi region, to detect all vectors, or to each of the fluorescent reporter genes (400 nM and 100 nM for primers and probe, respectively), DraI (40 U; New England Biolabs, Ipswich, MA), and 1.1 μl (4 μl for cfu) of the genomic DNA sample.

    Polymerase Chain Reaction:

    Article Title: SMN1 dosage analysis in spinal muscular atrophy from India
    Article Snippet: Each patient sample was analyzed in duplicate, and controls included 5 known two-copy, a one-copy and a homozygous deleted DNA samples, as well as water (no DNA template control). .. After PCR, 4 μl of amplified DNA from each reaction was digested in a 6 μl reaction volume with 1 unit of Dra I (New England Biolabs, USA) for 3 hours at 37°C. .. Two μl of the Dra I digestion were mixed with 4 μl of formamide, 0.5 μl of Gene Scan-500 Rox (ABI) and 0.5 μl of loading buffer, heated at 95°C for 5 min and then placed on ice for 2 min. Digested PCR product mixtures were analyzed on an ABI 373 Genetic Analyzer instrument (ABI).

    Article Title: Disease progression in patients with single, large-scale mitochondrial DNA deletions
    Article Snippet: Long-range PCR products were further assessed by restriction digests to map the precise location of the mitochondrial DNA deletion breakpoints ( ). .. PCR amplimers (5 µl) were digested to a series of DNA fragments of known length and position within the mitochondrial genome using restriction enzymes XhoI, BamHI, XcmI and DraI (New England Biolabs) before separation through a 0.7% agarose gel. .. The size of restriction products allows the location of the mitochondrial DNA deletion within the genome to be estimated, guiding the choice of appropriate sequencing primers ( Supplementary Table 4 ) to characterize mitochondrial DNA deletion breakpoints by Sanger sequencing (BigDye® Terminator chemistries using an ABI 3130xl Genetic Analyser; Applied Biosystems).

    Article Title: Polymorphisms in thymidylate synthase gene and susceptibility to breast cancer in a Chinese population: a case-control analysis
    Article Snippet: For the TS 3'-UTR del6 polymorphism, 152 bp (i.e. del6) or 158 bp fragments (i.e. ins6) were amplified and then digested by Dra I (New England BioLabs, Inc., Beverly, MA). .. The variant (ins6) allele produces two fragments of 88 and 70 bp, while the wild-type (del6) allele produces a single 152-bp fragment.

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: Specifically, 1 μl of eluted asMIPs were added to a 50 μl PCR reaction containing 5'-biotinylated P1 and P2 (P1: bio-CGAACGACGA CATAGACCAC, P2: bio-CGCTTTAGGT GCAGACACAA) primers at 0.6 μM concentration and 1× Platinum PCR SuperMix (Invitrogen, catalog no. 11306-016). .. PCR reactions were carried out using the following three-step thermal profile: denaturation at 94°C for 15 seconds, annealing at 60°C for 1 minute and extension at 72°C for 5 s. The resulting PCR products were treated with 20 u of DraI (NEB catalog no. R0129S) incubated at 37°C for 1 hour; DraI digestion was used to separate the two labeled sequence tags, as well as remove the now extraneous interrogation sequences, which could confound hybridization. .. Unique primers were used to amplify asMIPs quantified using Illumina HTS; in all other respects the PCR reaction conditions were identical to those specified for microarray sample preparation.

    Article Title: Genetic Tests for Ecological and Allopatric Speciation in Anoles on an Island Archipelago
    Article Snippet: This enzyme distinguishes between the central lineage (uncut by this enzyme), the southern lineage (cut at position 598) and the clade comprising the southwestern and the northwestern lineages (cut at position 166). .. To further distinguish between southwest and northwest lineages, we digested the same fragment using the restriction enzyme DraI (New England Biolabs) that cuts the PCR products from the northwest lineage at position 227, while those from SW lineage were uncut by this enzyme. .. The habitat type at each site was estimated from a multivariate climatic profile using nineteen climatic variables from Worldclim ( http://www.worldclim.org/ ).

    Article Title: Association between two common polymorphisms (single nucleotide polymorphism -250G/A and -514C/T) of the hepatic lipase gene and coronary artery disease in type 2 diabetic patients
    Article Snippet: DNA was extracted from leukocytes, taken from peripheral blood, by using a salting-out procedure (Miller's method). .. [ ] Genotyping of -250G/A (rs2070895), and -514C/T (rs1800588) SNPs, in the promoter region of the LIPC gene were performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism, followed by digestion with the restriction enzymes DraI and NlaIII (New England Biolabs), respectively. .. Target DNA was amplified using two different primer pairs, one pair for -250G/A and the other for -514C/T polymorphism [ ].

    Article Title: The genetics of feed conversion efficiency traits in a commercial broiler line
    Article Snippet: In brief, genomic regions were amplified using primer combinations ggaAGK_f2/ggaAGK_r2 or ggaGTF2I_f1/ggaGTF2I_r1 (see ) in a standard PCR mix with SupraTherm Taq Polymerase (Genecraft, Lüdinghausen, Germany) as described elsewhere . .. Overnight digestion of amplification products was performed using 5 U/μl Dra I and Nsi I restriction enzymes (New England Biolabs, Frankfurt, Germany) for GTF2I SNP c.2011 A > C and AGK SNP c.1166 G > A, respectively.

    Article Title: Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice
    Article Snippet: Each PCR product was subjected to automatic electrophoresis using MultiNA. .. The PCR products were purified and analyzed by RFLP analyses using Nar I or Dra I (New England Biolabs Japan). .. The PCR products identified as positive were analyzed by direct sequencing.

    Article Title: Sema3d controls collective endothelial cell migration by distinct mechanisms via Nrp1 and PlxnD1
    Article Snippet: After DraI digestion (New England Biolabs, Inc.), gRNA was transcribed using MEGAshortscript T7 kit (Ambion). .. Nls-zCas9-nls mRNA was synthesized as previously described ( ).

    Article Title: MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4
    Article Snippet: For CD1 (NM_053056.2) and PAK4 (Variant 5, NM_001014834.2) full-length 3’ UTR luciferase reporter constructs were generated. .. PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA). .. The PAK4 constructs containing mutations at the sequence of binding region of potential binding sites were generated using a site directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA).

    Article Title: Mutations in monoamine oxidase (MAO) genes in mice lead to hypersensitivity to serotonin-enhancing drugs: implications for drug side effects in humans
    Article Snippet: PCR conditions were: 95 °C 15 min, (94 °C 30 s, 55 °C 30 s, 72 °C 1 min) ×35 cycles and 72 °C 5 min. .. The PCR product from the MAOA reaction was digested with Dra I (New England Biolabs, Ipswich, MA, USA) at 37 °C for 2 h. The amplified DNA product after Dra I digestion exhibits a 100- and 200-base pair (bp) product for WT mice, three bands of 100, 200 and 300 bps for heterozygous female mice and an uncut 300-bp product for MAOA mutant mice. .. For the MAOB reaction, primers were: 5′-CTACAAAGCA GATTGCCACGC-3′ and 5′-TACCTGACATCAACTGGTCCC-3′.

    Article Title: Evidence that RpoS (?S) in Borrelia burgdorferi Is Controlled Directly by RpoN (?54/?N)
    Article Snippet: The BD SMART IIA primer, which anneals to the BD SMART IIA oligonucleotide linked to the 3′ end of the first-strand cDNA, and an rpoS -specific primer upstream of the gene-specific primers used in first-strand cDNA synthesis (rpoSR125) (Table ) were used to amplify the resulting cDNAs. .. PCR-amplified inserts cloned into the pGEM-T Easy vector (Promega, Madison, WI) were digested with the restriction endonuclease DraI (New England Biolabs, Ipswich, MA) to verify that they represented rpoS . .. Seventeen of 25 clones contained rpoS -specific sequences, as determined by the DraI restriction pattern (not shown).

    Article Title: Parallel gene analysis with allele-specific padlock probes and tag microarrays
    Article Snippet: Genomic DNA was reduced in size by digestion using DraI and SspI (New England Biolabs) for 2 h in the recommended buffers. .. After ligation, 10 µl of exonuclease mix was added to the reactions for a final concentration of 67 mM Tris–HCl pH 9.0, 50 mM KCl, 6.7 mM MgCl2 , 0.05 µg/µl BSA, 0.35 U/µl exonuclease I (New England Biolabs) and 0.35 U/µl exonuclease III (Amersham Biosciences).

    Article Title: Differential microRNA epression in asthma and the role of miR-1248 in regulation of IL-5
    Article Snippet: To clone IL-5 3’UTR into the pmirGLO vector (Promega), the IL-5 3’UTR was amplified from total cDNA from PBMCs using the following primers: IL-5 3’UTR DraI forward: 5’-AAA TTT AAA AGA CTA AAC TGG TTT GTT GCA GC-3’; IL-5 3’UTR SalI reverse: 5’-AAA GTC GAC GAA CAG TTG TCT ATT TTT GTT TTA TTA GA-3’. .. The vector and PCR product were digested with DraI and SalI (both from New England Biolabs), gel purified, and ligated with Quick T4 DNA Ligase (New England Biolabs). .. Following transformation into NEB-5-α cells, samples were plated onto an ampicillin-containing agar plate, and colonies were selected and grown in 5 ml of Luria-Bertani broth.

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: The clonal N40 parent isolate (cN40) and each of the derived isolates were grown from frozen stocks at 34°C in BSK-H medium supplemented with 6% rabbit serum (Sigma Aldrich) to a density of ∼5*107 cells/ml and the genomic DNA was purified using the DNeasy Blood and Tissue Kit protocol for gram negative bacteria (Qiagen; Valencia, CA). .. The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. Long-range PCR amplification was conducted using the primers N40-vlsLR-R (5′ Phos - GCT GGA CTT GAA TTT GGT AGG GAT TC 3′ ) and N40-vlsLR-F (5′ Phos - GGT GAT GGT GCC GAT TCA AAA TCT GG 3′ ) which anneal to the unique conserved regions flanking the unexpressed cassettes.

    CRISPR:

    Article Title: Sema3d controls collective endothelial cell migration by distinct mechanisms via Nrp1 and PlxnD1
    Article Snippet: Paragraph title: Generation of sema3d mutants by CRISPR/Cas9-mediated mutagenesis ... After DraI digestion (New England Biolabs, Inc.), gRNA was transcribed using MEGAshortscript T7 kit (Ambion).

    Agarose Gel Electrophoresis:

    Article Title: Disease progression in patients with single, large-scale mitochondrial DNA deletions
    Article Snippet: Long-range PCR products were further assessed by restriction digests to map the precise location of the mitochondrial DNA deletion breakpoints ( ). .. PCR amplimers (5 µl) were digested to a series of DNA fragments of known length and position within the mitochondrial genome using restriction enzymes XhoI, BamHI, XcmI and DraI (New England Biolabs) before separation through a 0.7% agarose gel. .. The size of restriction products allows the location of the mitochondrial DNA deletion within the genome to be estimated, guiding the choice of appropriate sequencing primers ( Supplementary Table 4 ) to characterize mitochondrial DNA deletion breakpoints by Sanger sequencing (BigDye® Terminator chemistries using an ABI 3130xl Genetic Analyser; Applied Biosystems).

    Article Title: Genetic Tests for Ecological and Allopatric Speciation in Anoles on an Island Archipelago
    Article Snippet: The digested products were run on a 2% agarose gel containing ethidium bromide. .. To further distinguish between southwest and northwest lineages, we digested the same fragment using the restriction enzyme DraI (New England Biolabs) that cuts the PCR products from the northwest lineage at position 227, while those from SW lineage were uncut by this enzyme.

    Article Title: Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice
    Article Snippet: In some pups, multiple bands were detected by PCR; in these cases, each RFLP-positive band was cut out from the agarose gel, the DNA extracted and individually sequenced. .. The PCR products were purified and analyzed by RFLP analyses using Nar I or Dra I (New England Biolabs Japan).

    Mouse Assay:

    Article Title: Enrichment of Human Hematopoietic Stem/Progenitor Cells Facilitates Transduction for Stem Cell Gene Therapy
    Article Snippet: Paragraph title: Transplantation of Transduced Human CB CD34+ /CD38− Cells in Immune-Deficient Mice ... Reaction mixtures were prepared consisting of 22 μl volumes containing 1× ddPCR Master Mix (Bio-Rad, Hercules, CA), primers, and probe specific to either the HIV-1 Psi region, to detect all vectors, or to each of the fluorescent reporter genes (400 nM and 100 nM for primers and probe, respectively), DraI (40 U; New England Biolabs, Ipswich, MA), and 1.1 μl (4 μl for cfu) of the genomic DNA sample.

    Article Title: Mutations in monoamine oxidase (MAO) genes in mice lead to hypersensitivity to serotonin-enhancing drugs: implications for drug side effects in humans
    Article Snippet: PCR conditions were: 95 °C 15 min, (94 °C 30 s, 55 °C 30 s, 72 °C 1 min) ×35 cycles and 72 °C 5 min. .. The PCR product from the MAOA reaction was digested with Dra I (New England Biolabs, Ipswich, MA, USA) at 37 °C for 2 h. The amplified DNA product after Dra I digestion exhibits a 100- and 200-base pair (bp) product for WT mice, three bands of 100, 200 and 300 bps for heterozygous female mice and an uncut 300-bp product for MAOA mutant mice. .. For the MAOB reaction, primers were: 5′-CTACAAAGCA GATTGCCACGC-3′ and 5′-TACCTGACATCAACTGGTCCC-3′.

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: A clonal isolate of B. burgdorferi strain N40 was intradermally inoculated into three C3H/HeN mice and re-isolated after 12 months from the blood (derived isolate 1(36B), derived isolate 2(44B), and derived isolate 3(39B)) as previously described . .. The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B).

    Plasmid Preparation:

    Article Title: Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice
    Article Snippet: The PCR products were purified and analyzed by RFLP analyses using Nar I or Dra I (New England Biolabs Japan). .. The PCR products were purified and analyzed by RFLP analyses using Nar I or Dra I (New England Biolabs Japan).

    Article Title: Sema3d controls collective endothelial cell migration by distinct mechanisms via Nrp1 and PlxnD1
    Article Snippet: Annealed CRISPR target site primers (CRIsema3d SE, 5′-TAGGAGACAGGTCAGGACGGGA-3′, and AS, 5′-AAACTCCCGTCCTGACCTGTCT-3′) were cloned into the guide RNA (gRNA) expression vector pDR274 as previously described ( ). .. After DraI digestion (New England Biolabs, Inc.), gRNA was transcribed using MEGAshortscript T7 kit (Ambion).

    Article Title: MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4
    Article Snippet: For CD1 (NM_053056.2) and PAK4 (Variant 5, NM_001014834.2) full-length 3’ UTR luciferase reporter constructs were generated. .. PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA). .. The PAK4 constructs containing mutations at the sequence of binding region of potential binding sites were generated using a site directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA).

    Article Title: Evidence that RpoS (?S) in Borrelia burgdorferi Is Controlled Directly by RpoN (?54/?N)
    Article Snippet: The BD SMART IIA primer, which anneals to the BD SMART IIA oligonucleotide linked to the 3′ end of the first-strand cDNA, and an rpoS -specific primer upstream of the gene-specific primers used in first-strand cDNA synthesis (rpoSR125) (Table ) were used to amplify the resulting cDNAs. .. PCR-amplified inserts cloned into the pGEM-T Easy vector (Promega, Madison, WI) were digested with the restriction endonuclease DraI (New England Biolabs, Ipswich, MA) to verify that they represented rpoS . .. Seventeen of 25 clones contained rpoS -specific sequences, as determined by the DraI restriction pattern (not shown).

    Article Title: Differential microRNA epression in asthma and the role of miR-1248 in regulation of IL-5
    Article Snippet: To clone IL-5 3’UTR into the pmirGLO vector (Promega), the IL-5 3’UTR was amplified from total cDNA from PBMCs using the following primers: IL-5 3’UTR DraI forward: 5’-AAA TTT AAA AGA CTA AAC TGG TTT GTT GCA GC-3’; IL-5 3’UTR SalI reverse: 5’-AAA GTC GAC GAA CAG TTG TCT ATT TTT GTT TTA TTA GA-3’. .. The vector and PCR product were digested with DraI and SalI (both from New England Biolabs), gel purified, and ligated with Quick T4 DNA Ligase (New England Biolabs). .. Following transformation into NEB-5-α cells, samples were plated onto an ampicillin-containing agar plate, and colonies were selected and grown in 5 ml of Luria-Bertani broth.

    Article Title: Natural Selection Promotes Antigenic Evolvability
    Article Snippet: The clonal N40 parent isolate (cN40) and each of the derived isolates were grown from frozen stocks at 34°C in BSK-H medium supplemented with 6% rabbit serum (Sigma Aldrich) to a density of ∼5*107 cells/ml and the genomic DNA was purified using the DNeasy Blood and Tissue Kit protocol for gram negative bacteria (Qiagen; Valencia, CA). .. The vls cassette region from each isolate was cloned into BigEasy v2.0 Linear Cloning Kit (Lucigen; Middleton, WI) by either 1. ligating total genomic DNA treated with Mung Bean Nuclease and DraI (New England Biolabs (NEB); Beverly, MA) into the cloning vector (derived isolate 2(44B)) or 2. ligating a long-range PCR fragment containing the unexpressed cassettes into the cloning vector (isolates cN40, 36B, and 39B). .. Long-range PCR amplification was conducted using the primers N40-vlsLR-R (5′ Phos - GCT GGA CTT GAA TTT GGT AGG GAT TC 3′ ) and N40-vlsLR-F (5′ Phos - GGT GAT GGT GCC GAT TCA AAA TCT GG 3′ ) which anneal to the unique conserved regions flanking the unexpressed cassettes.

    Software:

    Article Title: SMN1 dosage analysis in spinal muscular atrophy from India
    Article Snippet: After PCR, 4 μl of amplified DNA from each reaction was digested in a 6 μl reaction volume with 1 unit of Dra I (New England Biolabs, USA) for 3 hours at 37°C. .. Two μl of the Dra I digestion were mixed with 4 μl of formamide, 0.5 μl of Gene Scan-500 Rox (ABI) and 0.5 μl of loading buffer, heated at 95°C for 5 min and then placed on ice for 2 min. Digested PCR product mixtures were analyzed on an ABI 373 Genetic Analyzer instrument (ABI).

    Electrophoresis:

    Article Title: Association between two common polymorphisms (single nucleotide polymorphism -250G/A and -514C/T) of the hepatic lipase gene and coronary artery disease in type 2 diabetic patients
    Article Snippet: [ ] Genotyping of -250G/A (rs2070895), and -514C/T (rs1800588) SNPs, in the promoter region of the LIPC gene were performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism, followed by digestion with the restriction enzymes DraI and NlaIII (New England Biolabs), respectively. .. Thermal cycling conditions were similar for two SNPs and were as follows: An initial denaturation step at 94°C for 5 min, 35 cycles at 94°C for 30 s, 59 or 57°C for 30 s (depend on primers), 72°C for 45 s and a final extension step at 72°C for 5 min.

    Article Title: Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice
    Article Snippet: Each PCR product was subjected to automatic electrophoresis using MultiNA. .. The PCR products were purified and analyzed by RFLP analyses using Nar I or Dra I (New England Biolabs Japan).

    Sample Prep:

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: Paragraph title: Microarray sample preparation ... PCR reactions were carried out using the following three-step thermal profile: denaturation at 94°C for 15 seconds, annealing at 60°C for 1 minute and extension at 72°C for 5 s. The resulting PCR products were treated with 20 u of DraI (NEB catalog no. R0129S) incubated at 37°C for 1 hour; DraI digestion was used to separate the two labeled sequence tags, as well as remove the now extraneous interrogation sequences, which could confound hybridization.

    In Vitro:

    Article Title: Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1
    Article Snippet: The in vitro MMR assays were carried out as described previously ( ). .. The reactions were terminated by a 30-min incubation with a stop solution (final concentrations: 0.5 mM EDTA, 1.5% SDS (sodium dodecyl sulfate), 2.5 mg/ml proteinase K), cleaned up on a MinElute column (Qiagen), and the recovered phagemid was subjected to restriction digest with 6 U SalI and 20 U DraI (NEB).

    Modification:

    Article Title: Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells
    Article Snippet: Paragraph title: DNA modification, 2D-AGE and Southern hybridisation ... 2.4 Total DNA was digested with DraI (New England Biolabs), and then precipitated and suspended in 10 mM Hepes-NaOH (pH 7.2).

    Quantitation Assay:

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: The specific process used for amplifying circularized asMIPs was tailored to the method of quantitation; for arrays, MIPs were amplified with biotinylated PCR primers before being hybridized to microarrays. .. PCR reactions were carried out using the following three-step thermal profile: denaturation at 94°C for 15 seconds, annealing at 60°C for 1 minute and extension at 72°C for 5 s. The resulting PCR products were treated with 20 u of DraI (NEB catalog no. R0129S) incubated at 37°C for 1 hour; DraI digestion was used to separate the two labeled sequence tags, as well as remove the now extraneous interrogation sequences, which could confound hybridization.

    Concentration Assay:

    Article Title: SMN1 dosage analysis in spinal muscular atrophy from India
    Article Snippet: After PCR, 4 μl of amplified DNA from each reaction was digested in a 6 μl reaction volume with 1 unit of Dra I (New England Biolabs, USA) for 3 hours at 37°C. .. After PCR, 4 μl of amplified DNA from each reaction was digested in a 6 μl reaction volume with 1 unit of Dra I (New England Biolabs, USA) for 3 hours at 37°C.

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: Specifically, 1 μl of eluted asMIPs were added to a 50 μl PCR reaction containing 5'-biotinylated P1 and P2 (P1: bio-CGAACGACGA CATAGACCAC, P2: bio-CGCTTTAGGT GCAGACACAA) primers at 0.6 μM concentration and 1× Platinum PCR SuperMix (Invitrogen, catalog no. 11306-016). .. PCR reactions were carried out using the following three-step thermal profile: denaturation at 94°C for 15 seconds, annealing at 60°C for 1 minute and extension at 72°C for 5 s. The resulting PCR products were treated with 20 u of DraI (NEB catalog no. R0129S) incubated at 37°C for 1 hour; DraI digestion was used to separate the two labeled sequence tags, as well as remove the now extraneous interrogation sequences, which could confound hybridization.

    Article Title: HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner
    Article Snippet: AlwNI , DraI and Mo-MuLV RT enzymes were purchased from New England Biolabs (Ipswich, MA). .. AlwNI , DraI and Mo-MuLV RT enzymes were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Parallel gene analysis with allele-specific padlock probes and tag microarrays
    Article Snippet: Genomic DNA was reduced in size by digestion using DraI and SspI (New England Biolabs) for 2 h in the recommended buffers. .. Aliquots of 10 µl of ligation reactions, containing 50 ng digested DNA or 2 µl of cDNA in 20 mM Tris–HCl pH 9.0, 100 mM KCl, 10 mM MgCl2 , 1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 400 mU Tth ligase or Ampligase (Epicentre) and 400 pM each padlock probe were placed in a thermal cycler at 95°C for 5 min, then cycled 20 times between 95°C for 2 min and 55°C for 20 min, followed by 95°C for 2 min. Homozygous genotypes that were not present among our samples were instead represented by synthetic 40mer oligonucleotide targets added at 45 zmol to separate ligation reactions.

    FACS:

    Article Title: Enrichment of Human Hematopoietic Stem/Progenitor Cells Facilitates Transduction for Stem Cell Gene Therapy
    Article Snippet: After antibody incubation, RBCs were lysed using BD FACS-Lysing Solution (BD Biosciences). .. Reaction mixtures were prepared consisting of 22 μl volumes containing 1× ddPCR Master Mix (Bio-Rad, Hercules, CA), primers, and probe specific to either the HIV-1 Psi region, to detect all vectors, or to each of the fluorescent reporter genes (400 nM and 100 nM for primers and probe, respectively), DraI (40 U; New England Biolabs, Ipswich, MA), and 1.1 μl (4 μl for cfu) of the genomic DNA sample.

    Variant Assay:

    Article Title: MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4
    Article Snippet: For CD1 (NM_053056.2) and PAK4 (Variant 5, NM_001014834.2) full-length 3’ UTR luciferase reporter constructs were generated. .. PCR amplified individual insert fragments were sub-cloned into a SacI and Xba1 or SacI and DraI (New England Bio Labs, Ipswich, MA, USA) digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI, USA).

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