gdna kit  (New England Biolabs)


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    Name:
    Monarch Genomic DNA Purification Kit
    Description:
    The Monarch Genomic DNA Purification Kit is a comprehensive solution for cell lysis RNA removal and purification of intact genomic DNA gDNA from a wide variety of biological samples including cultured cells blood and mammalian tissues Additionally bacteria and yeast can be processed with extra steps to enhance lysis in these tough to lyse samples Protocols are also included to enable purification from clinically relevant samples such as saliva and cheek swabs as well as rapid cleanup of previously extracted gDNA Purified gDNA has high quality metrics including A260 A280 1 8 and A260 A230 2 0 high DIN scores and minimal residual RNA The purified gDNA is suitable for downstream applications such as end point PCR qPCR and library prep for NGS It typically has a peak size of 50kb making this kit an excellent choice upstream of long read sequencing platforms
    Catalog Number:
    T3010L
    Price:
    395
    Category:
    Genomic DNA Purification Kits
    Size:
    150 preps
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    Structured Review

    New England Biolabs gdna kit
    Monarch Genomic DNA Purification Kit
    The Monarch Genomic DNA Purification Kit is a comprehensive solution for cell lysis RNA removal and purification of intact genomic DNA gDNA from a wide variety of biological samples including cultured cells blood and mammalian tissues Additionally bacteria and yeast can be processed with extra steps to enhance lysis in these tough to lyse samples Protocols are also included to enable purification from clinically relevant samples such as saliva and cheek swabs as well as rapid cleanup of previously extracted gDNA Purified gDNA has high quality metrics including A260 A280 1 8 and A260 A230 2 0 high DIN scores and minimal residual RNA The purified gDNA is suitable for downstream applications such as end point PCR qPCR and library prep for NGS It typically has a peak size of 50kb making this kit an excellent choice upstream of long read sequencing platforms
    https://www.bioz.com/result/gdna kit/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gdna kit - by Bioz Stars, 2021-06
    86/100 stars

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    Related Articles

    Amplification:

    Article Title: Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics
    Article Snippet: Briefly, for unmarked deletions, ∼1kb fragments immediately upstream and downstream of the target locus were cloned using Gibson assembly into the pMQ30 suicide vector , . .. Fragments amplified from P. aeruginosa PA14 genomic DNA (gDNA) and cleaned up using the Monarch PCR Purification kit (New England Biolabs) were used for Gibson assembly together with pMQ30 cut with SacI and HindIII. .. The assembled construct was then transformed into Escherichia coli DH10B, with transformants being selected in LB with 20 µg/mL gentamicin.

    Article Title: A High-Efficacy CRISPR Interference System for Gene Function Discovery in Zymomonas mobilis
    Article Snippet: Oligonucleotides (2 μM each) were annealed in 1× CutSmart buffer (NEB) at 95°C for 5 min, followed by cooling to room temperature. .. For method two, fragments were amplified by PCR with primers oJMP197 and oJMP198 from a 78-nt oligonucleotide, followed by digestion with BsaI-HF-v2 (catalog number R3733; NEB) and purification with the Monarch DNA purification kit (NEB) following the manufacturer’s oligonucleotide purification protocol. .. Inserts (2 μl of a 1:40 dilution of annealed oligonucleotides or 2 ng purified digested PCR product) were ligated into 50 ng BsaI-digested vector.

    Article Title: Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics
    Article Snippet: Briefly, for unmarked deletions, approximately 1-kb fragments immediately upstream and downstream of the target locus were cloned using Gibson assembly into the pMQ30 suicide vector [ , ]. .. Fragments amplified from P . aeruginosa PA14 genomic DNA (gDNA) and cleaned up using the Monarch PCR Purification kit (New England Biolabs, Ipswich, Massachusetts, USA) were used for Gibson assembly together with pMQ30 cut with SacI and HindIII. .. The assembled construct was then transformed into E . coli DH10B, with transformants being selected in LB with 20 μg/mL gentamicin.

    Polymerase Chain Reaction:

    Article Title: Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics
    Article Snippet: Briefly, for unmarked deletions, ∼1kb fragments immediately upstream and downstream of the target locus were cloned using Gibson assembly into the pMQ30 suicide vector , . .. Fragments amplified from P. aeruginosa PA14 genomic DNA (gDNA) and cleaned up using the Monarch PCR Purification kit (New England Biolabs) were used for Gibson assembly together with pMQ30 cut with SacI and HindIII. .. The assembled construct was then transformed into Escherichia coli DH10B, with transformants being selected in LB with 20 µg/mL gentamicin.

    Article Title: A High-Efficacy CRISPR Interference System for Gene Function Discovery in Zymomonas mobilis
    Article Snippet: Oligonucleotides (2 μM each) were annealed in 1× CutSmart buffer (NEB) at 95°C for 5 min, followed by cooling to room temperature. .. For method two, fragments were amplified by PCR with primers oJMP197 and oJMP198 from a 78-nt oligonucleotide, followed by digestion with BsaI-HF-v2 (catalog number R3733; NEB) and purification with the Monarch DNA purification kit (NEB) following the manufacturer’s oligonucleotide purification protocol. .. Inserts (2 μl of a 1:40 dilution of annealed oligonucleotides or 2 ng purified digested PCR product) were ligated into 50 ng BsaI-digested vector.

    Article Title: Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics
    Article Snippet: Briefly, for unmarked deletions, approximately 1-kb fragments immediately upstream and downstream of the target locus were cloned using Gibson assembly into the pMQ30 suicide vector [ , ]. .. Fragments amplified from P . aeruginosa PA14 genomic DNA (gDNA) and cleaned up using the Monarch PCR Purification kit (New England Biolabs, Ipswich, Massachusetts, USA) were used for Gibson assembly together with pMQ30 cut with SacI and HindIII. .. The assembled construct was then transformed into E . coli DH10B, with transformants being selected in LB with 20 μg/mL gentamicin.

    Purification:

    Article Title: Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics
    Article Snippet: Briefly, for unmarked deletions, ∼1kb fragments immediately upstream and downstream of the target locus were cloned using Gibson assembly into the pMQ30 suicide vector , . .. Fragments amplified from P. aeruginosa PA14 genomic DNA (gDNA) and cleaned up using the Monarch PCR Purification kit (New England Biolabs) were used for Gibson assembly together with pMQ30 cut with SacI and HindIII. .. The assembled construct was then transformed into Escherichia coli DH10B, with transformants being selected in LB with 20 µg/mL gentamicin.

    Article Title: A High-Efficacy CRISPR Interference System for Gene Function Discovery in Zymomonas mobilis
    Article Snippet: Oligonucleotides (2 μM each) were annealed in 1× CutSmart buffer (NEB) at 95°C for 5 min, followed by cooling to room temperature. .. For method two, fragments were amplified by PCR with primers oJMP197 and oJMP198 from a 78-nt oligonucleotide, followed by digestion with BsaI-HF-v2 (catalog number R3733; NEB) and purification with the Monarch DNA purification kit (NEB) following the manufacturer’s oligonucleotide purification protocol. .. Inserts (2 μl of a 1:40 dilution of annealed oligonucleotides or 2 ng purified digested PCR product) were ligated into 50 ng BsaI-digested vector.

    Article Title: Direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping (CasKAS)
    Article Snippet: Cells were incubated for 10 minutes at 37 ° C with shaking at 500 rpm in a Thermomixer. .. Cells were then pelleted by centrifuging at 500 g for 5 minutes at 4 ° C. Genomic DNA was then extracted using the Monarch gDNA Purification Kit (NEB T3010S) following the standard protocol but with elution using 175 µ L 25 mM K3 BO3 at pH 7.0. .. Click reaction, biotin pull down and library generation The click reaction was carried out by combining 175 µ L purified DNA, 5 µ L 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma 760749), and 20 µ L 10×PBS in a final volume of 200 µ L or 87.5 µ L purified and sheared DNA, 2.5 µ L 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma 760749), and 10 µ L 10×PBS in a final volume of 100 µ L. The reaction was incubated at 37 °C for 90 minutes.

    Article Title: Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics
    Article Snippet: Briefly, for unmarked deletions, approximately 1-kb fragments immediately upstream and downstream of the target locus were cloned using Gibson assembly into the pMQ30 suicide vector [ , ]. .. Fragments amplified from P . aeruginosa PA14 genomic DNA (gDNA) and cleaned up using the Monarch PCR Purification kit (New England Biolabs, Ipswich, Massachusetts, USA) were used for Gibson assembly together with pMQ30 cut with SacI and HindIII. .. The assembled construct was then transformed into E . coli DH10B, with transformants being selected in LB with 20 μg/mL gentamicin.

    Infection:

    Article Title: MicroRNA-21 depletion by CRISPR/Cas9 in CNE2 nasopharyngeal cells hinders proliferation and induces apoptosis by targeting the PI3K/AKT/MOTOR signaling pathway
    Article Snippet: The oligonucleotide sequence was synthesized by Shanghai Genechem Co., LTD., and each pair of oligonucleotides was transformed into DS-DNA fragments through annealing and then linked onto the Cas9-Easy-lentivirus vector. .. The CNE2 cells were collected after 72 h of infection, along with the application of mammalian genomic DNA extraction kit to extract genome DNA and T7EN1 (NEB, USA; M0302S) to detect the editing efficiency of gRNAs. .. The whole genomic DNA was extracted by utilizing the DNA Extraction kit (Beyotime, Shaihai, China) reported previously [ ].

    DNA Extraction:

    Article Title: MicroRNA-21 depletion by CRISPR/Cas9 in CNE2 nasopharyngeal cells hinders proliferation and induces apoptosis by targeting the PI3K/AKT/MOTOR signaling pathway
    Article Snippet: The oligonucleotide sequence was synthesized by Shanghai Genechem Co., LTD., and each pair of oligonucleotides was transformed into DS-DNA fragments through annealing and then linked onto the Cas9-Easy-lentivirus vector. .. The CNE2 cells were collected after 72 h of infection, along with the application of mammalian genomic DNA extraction kit to extract genome DNA and T7EN1 (NEB, USA; M0302S) to detect the editing efficiency of gRNAs. .. The whole genomic DNA was extracted by utilizing the DNA Extraction kit (Beyotime, Shaihai, China) reported previously [ ].

    Mutagenesis:

    Article Title: The Borrelia burgdorferi Glycosaminoglycan Binding Protein Bgp in the B31 Strain Is Not Essential for Infectivity despite Facilitating Adherence and Tissue Colonization
    Article Snippet: .. Genomic DNA from the bgp mutant strain was isolated by standard genomic DNA purification, digested with the restriction enzyme Spe1 (New England BioLabs, Ipswich, MA), and cloned in pUC19 (Thermo Scientific, Waltham, MA). .. Transformation of Top10 competent E. coli cells was followed by selection on gentamicin (15 μg/ml)-containing Luria-Bertani plates to select for the DNA fragment containing the resistance cassette of the transposon.

    Isolation:

    Article Title: The Borrelia burgdorferi Glycosaminoglycan Binding Protein Bgp in the B31 Strain Is Not Essential for Infectivity despite Facilitating Adherence and Tissue Colonization
    Article Snippet: .. Genomic DNA from the bgp mutant strain was isolated by standard genomic DNA purification, digested with the restriction enzyme Spe1 (New England BioLabs, Ipswich, MA), and cloned in pUC19 (Thermo Scientific, Waltham, MA). .. Transformation of Top10 competent E. coli cells was followed by selection on gentamicin (15 μg/ml)-containing Luria-Bertani plates to select for the DNA fragment containing the resistance cassette of the transposon.

    DNA Purification:

    Article Title: The Borrelia burgdorferi Glycosaminoglycan Binding Protein Bgp in the B31 Strain Is Not Essential for Infectivity despite Facilitating Adherence and Tissue Colonization
    Article Snippet: .. Genomic DNA from the bgp mutant strain was isolated by standard genomic DNA purification, digested with the restriction enzyme Spe1 (New England BioLabs, Ipswich, MA), and cloned in pUC19 (Thermo Scientific, Waltham, MA). .. Transformation of Top10 competent E. coli cells was followed by selection on gentamicin (15 μg/ml)-containing Luria-Bertani plates to select for the DNA fragment containing the resistance cassette of the transposon.

    Article Title: A High-Efficacy CRISPR Interference System for Gene Function Discovery in Zymomonas mobilis
    Article Snippet: Oligonucleotides (2 μM each) were annealed in 1× CutSmart buffer (NEB) at 95°C for 5 min, followed by cooling to room temperature. .. For method two, fragments were amplified by PCR with primers oJMP197 and oJMP198 from a 78-nt oligonucleotide, followed by digestion with BsaI-HF-v2 (catalog number R3733; NEB) and purification with the Monarch DNA purification kit (NEB) following the manufacturer’s oligonucleotide purification protocol. .. Inserts (2 μl of a 1:40 dilution of annealed oligonucleotides or 2 ng purified digested PCR product) were ligated into 50 ng BsaI-digested vector.

    Article Title: A New Protocol for Targeted insertion using CRISPR-Cas9, Oligo Single-Stranded DNA and Protoplast Regeneration
    Article Snippet: .. Whole genome sequencing for off-targeted insertion analysis Leaves of N. benthamiana protoplast regenerants were collected for Genomic DNA purification. .. Genomic DNA for genome sequencing was extracted using a Plant DNA Purification Kit (DP320, Tiangen, http://www.tiangen.com/en/ ).

    Clone Assay:

    Article Title: The Borrelia burgdorferi Glycosaminoglycan Binding Protein Bgp in the B31 Strain Is Not Essential for Infectivity despite Facilitating Adherence and Tissue Colonization
    Article Snippet: .. Genomic DNA from the bgp mutant strain was isolated by standard genomic DNA purification, digested with the restriction enzyme Spe1 (New England BioLabs, Ipswich, MA), and cloned in pUC19 (Thermo Scientific, Waltham, MA). .. Transformation of Top10 competent E. coli cells was followed by selection on gentamicin (15 μg/ml)-containing Luria-Bertani plates to select for the DNA fragment containing the resistance cassette of the transposon.

    Sequencing:

    Article Title: A New Protocol for Targeted insertion using CRISPR-Cas9, Oligo Single-Stranded DNA and Protoplast Regeneration
    Article Snippet: .. Whole genome sequencing for off-targeted insertion analysis Leaves of N. benthamiana protoplast regenerants were collected for Genomic DNA purification. .. Genomic DNA for genome sequencing was extracted using a Plant DNA Purification Kit (DP320, Tiangen, http://www.tiangen.com/en/ ).

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    New England Biolabs monarch pcr purification kit
    PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. (A) Structures of PYO, 2 representative fluoroquinolones (CIP and LVX) and 2 representative aminoglycosides (GEN and TOB). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not [ 21 , 22 ]. Rings with an aromatic character are highlighted in red. (B) Normalized cDNA levels for genes within operons coding for the 11 main RND efflux systems in P . <t>aeruginosa</t> (left; n = 3) and PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI-opmD systems (right; n = 3). For full <t>qRT-PCR</t> dataset, see S1 – S3 Figs. (C) Effect of PYO on tolerance to CIP (1 μg/mL), LVX (1 μg/mL), and CST (16 μg/mL) in GMM ( n = 4). (D) Effect of PYO on tolerance to CIP (1 μg/mL) and TOB (40 μg/mL) in SCFM ( n = 4). PYO itself was not toxic under the experimental conditions [ 16 ] ( S4C Fig ). WT made 50–80 μM PYO as measured by absorbance of the culture supernatant at 691 nm. See S5A Fig for experimental design. (E) Effect on tolerance to CIP (1 μg/mL) in GMM caused by the presence of the 4 main phenazines produced by P . aeruginosa (PYO, PCA, PCN, and 1-OH-PHZ) ( n = 4). For this experiment, a Δ phz * strain that cannot produce or modify any phenazine was used (see Methods ). (F, G) Effect of PYO on lag during outgrowth after exposure to CIP in GMM. A representative field of view over different time points (F; magenta = WT::mApple, green = Δ phz ::GFP; see S1 Movie ) is shown together with the quantification of growth area on the agarose pads at time 0 hour and 15 hours (G). For these experiments, a culture of each strain tested was grown and exposed to CIP (10 μg/mL) separately, then cells of both cultures were washed, mixed, and placed together on a pad and imaged during outgrowth. The pads did not contain any PYO or CIP (see Methods and S5D Fig for details). White arrows in the displayed images point to regions with faster recovery of WT growth. The field of view displayed is marked with a black arrow in the quantification plot. The results for the experiment with swapped fluorescent proteins are shown in S4E Fig . See S4C Fig for complementary data about effects of PYO on lag. Scale bar: 20 μm. (H) Tolerance of Δ phz to CIP (1 μg/mL) in GMM in the presence of different concentrations of PYO ( n = 4). (G) Tolerance of Δ phz to CIP (1 μg/mL) in GMM upon artificial induction of the mexGHI-opmD operon with arabinose ( n = 4). The dashed green line marks the average survival of PYO-producing WT under similar conditions (without arabinose). Statistics: C, D, E, H—1-way ANOVA with Tukey HSD multiple comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); G, I—Welch unpaired t test (* p
    Monarch Pcr Purification Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch pcr purification kit/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monarch pcr purification kit - by Bioz Stars, 2021-06
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    PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. (A) Structures of PYO, 2 representative fluoroquinolones (CIP and LVX) and 2 representative aminoglycosides (GEN and TOB). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not [ 21 , 22 ]. Rings with an aromatic character are highlighted in red. (B) Normalized cDNA levels for genes within operons coding for the 11 main RND efflux systems in P . aeruginosa (left; n = 3) and PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI-opmD systems (right; n = 3). For full qRT-PCR dataset, see S1 – S3 Figs. (C) Effect of PYO on tolerance to CIP (1 μg/mL), LVX (1 μg/mL), and CST (16 μg/mL) in GMM ( n = 4). (D) Effect of PYO on tolerance to CIP (1 μg/mL) and TOB (40 μg/mL) in SCFM ( n = 4). PYO itself was not toxic under the experimental conditions [ 16 ] ( S4C Fig ). WT made 50–80 μM PYO as measured by absorbance of the culture supernatant at 691 nm. See S5A Fig for experimental design. (E) Effect on tolerance to CIP (1 μg/mL) in GMM caused by the presence of the 4 main phenazines produced by P . aeruginosa (PYO, PCA, PCN, and 1-OH-PHZ) ( n = 4). For this experiment, a Δ phz * strain that cannot produce or modify any phenazine was used (see Methods ). (F, G) Effect of PYO on lag during outgrowth after exposure to CIP in GMM. A representative field of view over different time points (F; magenta = WT::mApple, green = Δ phz ::GFP; see S1 Movie ) is shown together with the quantification of growth area on the agarose pads at time 0 hour and 15 hours (G). For these experiments, a culture of each strain tested was grown and exposed to CIP (10 μg/mL) separately, then cells of both cultures were washed, mixed, and placed together on a pad and imaged during outgrowth. The pads did not contain any PYO or CIP (see Methods and S5D Fig for details). White arrows in the displayed images point to regions with faster recovery of WT growth. The field of view displayed is marked with a black arrow in the quantification plot. The results for the experiment with swapped fluorescent proteins are shown in S4E Fig . See S4C Fig for complementary data about effects of PYO on lag. Scale bar: 20 μm. (H) Tolerance of Δ phz to CIP (1 μg/mL) in GMM in the presence of different concentrations of PYO ( n = 4). (G) Tolerance of Δ phz to CIP (1 μg/mL) in GMM upon artificial induction of the mexGHI-opmD operon with arabinose ( n = 4). The dashed green line marks the average survival of PYO-producing WT under similar conditions (without arabinose). Statistics: C, D, E, H—1-way ANOVA with Tukey HSD multiple comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); G, I—Welch unpaired t test (* p

    Journal: PLoS Biology

    Article Title: Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics

    doi: 10.1371/journal.pbio.3001093

    Figure Lengend Snippet: PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. (A) Structures of PYO, 2 representative fluoroquinolones (CIP and LVX) and 2 representative aminoglycosides (GEN and TOB). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not [ 21 , 22 ]. Rings with an aromatic character are highlighted in red. (B) Normalized cDNA levels for genes within operons coding for the 11 main RND efflux systems in P . aeruginosa (left; n = 3) and PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI-opmD systems (right; n = 3). For full qRT-PCR dataset, see S1 – S3 Figs. (C) Effect of PYO on tolerance to CIP (1 μg/mL), LVX (1 μg/mL), and CST (16 μg/mL) in GMM ( n = 4). (D) Effect of PYO on tolerance to CIP (1 μg/mL) and TOB (40 μg/mL) in SCFM ( n = 4). PYO itself was not toxic under the experimental conditions [ 16 ] ( S4C Fig ). WT made 50–80 μM PYO as measured by absorbance of the culture supernatant at 691 nm. See S5A Fig for experimental design. (E) Effect on tolerance to CIP (1 μg/mL) in GMM caused by the presence of the 4 main phenazines produced by P . aeruginosa (PYO, PCA, PCN, and 1-OH-PHZ) ( n = 4). For this experiment, a Δ phz * strain that cannot produce or modify any phenazine was used (see Methods ). (F, G) Effect of PYO on lag during outgrowth after exposure to CIP in GMM. A representative field of view over different time points (F; magenta = WT::mApple, green = Δ phz ::GFP; see S1 Movie ) is shown together with the quantification of growth area on the agarose pads at time 0 hour and 15 hours (G). For these experiments, a culture of each strain tested was grown and exposed to CIP (10 μg/mL) separately, then cells of both cultures were washed, mixed, and placed together on a pad and imaged during outgrowth. The pads did not contain any PYO or CIP (see Methods and S5D Fig for details). White arrows in the displayed images point to regions with faster recovery of WT growth. The field of view displayed is marked with a black arrow in the quantification plot. The results for the experiment with swapped fluorescent proteins are shown in S4E Fig . See S4C Fig for complementary data about effects of PYO on lag. Scale bar: 20 μm. (H) Tolerance of Δ phz to CIP (1 μg/mL) in GMM in the presence of different concentrations of PYO ( n = 4). (G) Tolerance of Δ phz to CIP (1 μg/mL) in GMM upon artificial induction of the mexGHI-opmD operon with arabinose ( n = 4). The dashed green line marks the average survival of PYO-producing WT under similar conditions (without arabinose). Statistics: C, D, E, H—1-way ANOVA with Tukey HSD multiple comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); G, I—Welch unpaired t test (* p

    Article Snippet: Fragments amplified from P . aeruginosa PA14 genomic DNA (gDNA) and cleaned up using the Monarch PCR Purification kit (New England Biolabs, Ipswich, Massachusetts, USA) were used for Gibson assembly together with pMQ30 cut with SacI and HindIII.

    Techniques: Expressing, Quantitative RT-PCR, Produced

    Mobile-CRISPRi system for transcriptional repression optimized for Zymomonas mobilis . (A) Modular Z. mobilis CRISPRi system encodes dCas9, sgRNA, and antibiotic resistance cassettes on a Tn 7 transposon. The promoter (P 1 ) and ribosome binding site (rbs) for dCas9 and the promoter (P C ) for the sgRNA have been optimized for Z. mobilis . DNA encoding the 20-nt variable region of the sgRNA can be cloned (individually or libraries) in between the BsaI sites. (B) CRISPRi-expressing strains are constructed by triparental mating of E. coli donor strains (one harboring the Mobile-CRISPRi plasmid and another harboring a plasmid expressing the Tn 7 transposase) with Z. mobilis . The CRISPRi expression cassette will be stably incorporated onto the Z. mobilis chromosome at the Tn 7 att site located downstream from glmS . (C) Optimization of sgRNA expression. Six promoter sequences (A to F) based on either lacUV5 or a synthetic promoter were incorporated into the CRISPRi system. Alignment is to the E. coli σ 70 consensus promoter, with the −10 and −35 core promoter elements underlined and shown in boldface, the lac operator locations highlighted in green or cyan, and the UP element highlighted in orange. (D) Comparison of Z. mobilis Mobile-CRISPRi sgRNA promoter variants. A GFP expression cassette was cloned into the PmeI site, and an sgRNA targeting GFP (or a nontargeting control) was cloned into the BsaI sites. Cultures were diluted 1:1,000 and incubated in medium with 0 or 1 mM IPTG for ∼10 doublings prior to measurement of GFP expression. Expression was normalized to the results for a non GFP-expressing strain. Standard deviations of the results from 4 biological replicates are shown. (E) Expression of Z. mobilis CRISPRi system is inducible over a range of IPTG concentrations. Standard deviations are shown.

    Journal: Applied and Environmental Microbiology

    Article Title: A High-Efficacy CRISPR Interference System for Gene Function Discovery in Zymomonas mobilis

    doi: 10.1128/AEM.01621-20

    Figure Lengend Snippet: Mobile-CRISPRi system for transcriptional repression optimized for Zymomonas mobilis . (A) Modular Z. mobilis CRISPRi system encodes dCas9, sgRNA, and antibiotic resistance cassettes on a Tn 7 transposon. The promoter (P 1 ) and ribosome binding site (rbs) for dCas9 and the promoter (P C ) for the sgRNA have been optimized for Z. mobilis . DNA encoding the 20-nt variable region of the sgRNA can be cloned (individually or libraries) in between the BsaI sites. (B) CRISPRi-expressing strains are constructed by triparental mating of E. coli donor strains (one harboring the Mobile-CRISPRi plasmid and another harboring a plasmid expressing the Tn 7 transposase) with Z. mobilis . The CRISPRi expression cassette will be stably incorporated onto the Z. mobilis chromosome at the Tn 7 att site located downstream from glmS . (C) Optimization of sgRNA expression. Six promoter sequences (A to F) based on either lacUV5 or a synthetic promoter were incorporated into the CRISPRi system. Alignment is to the E. coli σ 70 consensus promoter, with the −10 and −35 core promoter elements underlined and shown in boldface, the lac operator locations highlighted in green or cyan, and the UP element highlighted in orange. (D) Comparison of Z. mobilis Mobile-CRISPRi sgRNA promoter variants. A GFP expression cassette was cloned into the PmeI site, and an sgRNA targeting GFP (or a nontargeting control) was cloned into the BsaI sites. Cultures were diluted 1:1,000 and incubated in medium with 0 or 1 mM IPTG for ∼10 doublings prior to measurement of GFP expression. Expression was normalized to the results for a non GFP-expressing strain. Standard deviations of the results from 4 biological replicates are shown. (E) Expression of Z. mobilis CRISPRi system is inducible over a range of IPTG concentrations. Standard deviations are shown.

    Article Snippet: For method two, fragments were amplified by PCR with primers oJMP197 and oJMP198 from a 78-nt oligonucleotide, followed by digestion with BsaI-HF-v2 (catalog number R3733; NEB) and purification with the Monarch DNA purification kit (NEB) following the manufacturer’s oligonucleotide purification protocol.

    Techniques: Binding Assay, Clone Assay, Expressing, Construct, Plasmid Preparation, Stable Transfection, Incubation

    CRISPR/cas9 can significantly downregulate the expression of miR-21 in CNE2 cells. A. Schematic diagram of the design of sgRNAs for miR-21 . B. DNA cleavage by CRISPR/cas9 is detected by T7EN1 assay. C. Design of the primers of miR-21 in the genome. D. Expression of mature miR-21 following CRISPR/cas9 editing by PCR.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: MicroRNA-21 depletion by CRISPR/Cas9 in CNE2 nasopharyngeal cells hinders proliferation and induces apoptosis by targeting the PI3K/AKT/MOTOR signaling pathway

    doi:

    Figure Lengend Snippet: CRISPR/cas9 can significantly downregulate the expression of miR-21 in CNE2 cells. A. Schematic diagram of the design of sgRNAs for miR-21 . B. DNA cleavage by CRISPR/cas9 is detected by T7EN1 assay. C. Design of the primers of miR-21 in the genome. D. Expression of mature miR-21 following CRISPR/cas9 editing by PCR.

    Article Snippet: The CNE2 cells were collected after 72 h of infection, along with the application of mammalian genomic DNA extraction kit to extract genome DNA and T7EN1 (NEB, USA; M0302S) to detect the editing efficiency of gRNAs.

    Techniques: CRISPR, Expressing, Polymerase Chain Reaction

    Growth and bgp expression in strains B31 5A4 (WT), bgp mut, and bgp comp. (A) Reverse transcriptase PCR (RT-PCR) with bgp-pta operon primers produced a 1.9-kb product in WT and bgp comp strains. No transcript was detected in the bgp mut strain. RT-PCR using pta primers produced a 1-kb transcript, indicating that transcription of the downstream gene was not affected by the bgp mutagenesis. A control without reverse transcriptase indicates that all RNA samples were free from DNA contamination. (B) Growth rates of all B. burgdorferi strains were comparable. (C) Western blot analysis using anti-Bgp antibodies showed the absence of Bgp expression in the mutant strain. Anti-Pta antibodies showed that this protein is expressed uniformly, albeit at low levels, in all strains. Flagellin protein detected by anti-FlaB antibodies was included as a loading control.

    Journal: Infection and Immunity

    Article Title: The Borrelia burgdorferi Glycosaminoglycan Binding Protein Bgp in the B31 Strain Is Not Essential for Infectivity despite Facilitating Adherence and Tissue Colonization

    doi: 10.1128/IAI.00667-17

    Figure Lengend Snippet: Growth and bgp expression in strains B31 5A4 (WT), bgp mut, and bgp comp. (A) Reverse transcriptase PCR (RT-PCR) with bgp-pta operon primers produced a 1.9-kb product in WT and bgp comp strains. No transcript was detected in the bgp mut strain. RT-PCR using pta primers produced a 1-kb transcript, indicating that transcription of the downstream gene was not affected by the bgp mutagenesis. A control without reverse transcriptase indicates that all RNA samples were free from DNA contamination. (B) Growth rates of all B. burgdorferi strains were comparable. (C) Western blot analysis using anti-Bgp antibodies showed the absence of Bgp expression in the mutant strain. Anti-Pta antibodies showed that this protein is expressed uniformly, albeit at low levels, in all strains. Flagellin protein detected by anti-FlaB antibodies was included as a loading control.

    Article Snippet: Genomic DNA from the bgp mutant strain was isolated by standard genomic DNA purification, digested with the restriction enzyme Spe1 (New England BioLabs, Ipswich, MA), and cloned in pUC19 (Thermo Scientific, Waltham, MA).

    Techniques: Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Produced, Mutagenesis, Western Blot