monarch pcr purification 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 monarch pcr purification 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/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, 2021-05
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    Images

    1) Product Images from "Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics"

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.049437

    PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. A . Structures of PYO, two representative fluoroquinolones (CP = ciprofloxacin, LV = levofloxacin) and two representative aminoglycosides (GM = gentamicin, TM = tobramycin). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not 11 . 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). PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI- opmD systems (right; n = 3). For full qRT-PCR dataset, see Figs. S2, S3 and S4. C . Effect of PYO on tolerance to CP and LV in glucose minimal medium (left), and to CP in SCFM (right) (all 1 µg/mL) (n = 4). PYO itself was not toxic under the experimental conditions 8 . WT made 50-80 µM PYO as measured by absorbance of the culture supernatant at 691 nm. See Fig. S5A for experimental design. D-E . Effect of PYO on lag during outgrowth after exposure to CP. A representative field of view over different time points (D; magenta = WT::mApple, green = Δ phz ::GFP; see Movie S1) is shown together with the quantification of growth area on the agarose pads at time 0 hrs and 15 hrs (E). For these experiments, a culture of each strain tested was grown and exposed to CP (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 CP (see Methods and Fig. S5D 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 Fig. S5E. Scale bar: 20 µm. F . Tolerance of Δ phz to CP (1 µg/mL) in stationary phase in the presence of different concentrations of PYO (n = 4). G . Tolerance of Δ phz to CP (1 µg/mL) 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, F – One-way ANOVA with Tukey’s HSD multiple-comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); E, G – Welch’s unpaired t- test (* p
    Figure Legend Snippet: PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. A . Structures of PYO, two representative fluoroquinolones (CP = ciprofloxacin, LV = levofloxacin) and two representative aminoglycosides (GM = gentamicin, TM = tobramycin). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not 11 . 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). PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI- opmD systems (right; n = 3). For full qRT-PCR dataset, see Figs. S2, S3 and S4. C . Effect of PYO on tolerance to CP and LV in glucose minimal medium (left), and to CP in SCFM (right) (all 1 µg/mL) (n = 4). PYO itself was not toxic under the experimental conditions 8 . WT made 50-80 µM PYO as measured by absorbance of the culture supernatant at 691 nm. See Fig. S5A for experimental design. D-E . Effect of PYO on lag during outgrowth after exposure to CP. A representative field of view over different time points (D; magenta = WT::mApple, green = Δ phz ::GFP; see Movie S1) is shown together with the quantification of growth area on the agarose pads at time 0 hrs and 15 hrs (E). For these experiments, a culture of each strain tested was grown and exposed to CP (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 CP (see Methods and Fig. S5D 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 Fig. S5E. Scale bar: 20 µm. F . Tolerance of Δ phz to CP (1 µg/mL) in stationary phase in the presence of different concentrations of PYO (n = 4). G . Tolerance of Δ phz to CP (1 µg/mL) 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, F – One-way ANOVA with Tukey’s HSD multiple-comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); E, G – Welch’s unpaired t- test (* p

    Techniques Used: Expressing, Quantitative RT-PCR

    2) Product Images from "Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe"

    Article Title: Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe

    Journal: bioRxiv

    doi: 10.1101/2021.04.12.437332

    Upf1 association to specific genes is RNase sensitive A ) IGB screenshot of ChIP-chip enrichments of Upf1-HA in asynchronous culture (top row) and in S-phase culture (bottom row) over act1 gene and its flanking region. Genes and genomic features are shown below. B ) Top panel-schematic diagram of the act1 gene with CDS sequence (in grey); the PCR amplicons used for the ChIP assay are indicated by the dotted lines above (numbers correspond to the primer positions relative to start codon). Bottom panel-polyacrylamide gels showing radiolabelled PCR products produced by the act1 specific primer pairs (top bands) and by the pair specific for the intergenic region (bottom bands); using input DNA before ChIP (left panel) and using ChIP-enriched DNA from asynchronous (middle panel) and S-phase culture (right panel). The relative enrichment of act1 DNA relative to intergenic sequence is expressed as a ratio of the intensity of the same fragments produced with the input DNA. C ) Independent qPCR quantification of Upf1-Flag ChIP signal on 4 specific regions of pma1 gene and 1 intergenic control in the absence and presence of RNase. N.T-non-tagged.
    Figure Legend Snippet: Upf1 association to specific genes is RNase sensitive A ) IGB screenshot of ChIP-chip enrichments of Upf1-HA in asynchronous culture (top row) and in S-phase culture (bottom row) over act1 gene and its flanking region. Genes and genomic features are shown below. B ) Top panel-schematic diagram of the act1 gene with CDS sequence (in grey); the PCR amplicons used for the ChIP assay are indicated by the dotted lines above (numbers correspond to the primer positions relative to start codon). Bottom panel-polyacrylamide gels showing radiolabelled PCR products produced by the act1 specific primer pairs (top bands) and by the pair specific for the intergenic region (bottom bands); using input DNA before ChIP (left panel) and using ChIP-enriched DNA from asynchronous (middle panel) and S-phase culture (right panel). The relative enrichment of act1 DNA relative to intergenic sequence is expressed as a ratio of the intensity of the same fragments produced with the input DNA. C ) Independent qPCR quantification of Upf1-Flag ChIP signal on 4 specific regions of pma1 gene and 1 intergenic control in the absence and presence of RNase. N.T-non-tagged.

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, Polymerase Chain Reaction, Produced, Real-time Polymerase Chain Reaction

    Genome-wide association of Upf1 with protein coding genes A) IGB visualisation of Upf1-HA ChIP-chip enrichment in asynchronous culture (top track, shown in red) and in S-phase culture (bottom track, shown in sky blue) at a representative chromosomal region (270 kb) of S. pombe indicating enrichment of several specific genes, those discussed in the main text are labelled. Genes and genomic features are shown in black below. B ) Zoomed-in view of Upf1 enrichment over the entire pma1 gene (highlighted in dark blue) - 5’UTR (in grey), CDS (in purple) and 3’UTR (in grey) of pma1 are shown in the bottom row schematic. C ) Top panel-diagram of the pma1 gene (cDNA region in grey) and the positions of amplicons used for the PCR-ChIP assay are indicated by the dotted lines above (numbers correspond to the position of the primers relative to start codon). Bottom panel-polyacrylamide gels showing radiolabelled PCR products produced by the pma1 -specific primer pairs (top bands) and by the intergenic region specific primer (bottom bands, labelled Int.); using input DNA before ChIP (left panel) and using ChIP-enriched DNA from asynchronous culture without (middle panel) and with RNase pre-treatment of the chromatin (right panel). The relative enrichment of pma1 DNA relative to intergenic sequence is expressed as a ratio of the intensity of the same fragments produced with the input DNA. D ) PCR analysis as in C using input DNA before ChIP (left panel) and using ChIP-enriched DNA from S-phase culture without the RNase treatment (right panel). E ) Pie-charts showing the proportion of different classes of gene associated to Upf1 in asynchronous and S-phase culture of S. pombe. F ) ChIP-qPCR of Upf1-Flag enrichment over pma1 in three independent asynchronous cultures.
    Figure Legend Snippet: Genome-wide association of Upf1 with protein coding genes A) IGB visualisation of Upf1-HA ChIP-chip enrichment in asynchronous culture (top track, shown in red) and in S-phase culture (bottom track, shown in sky blue) at a representative chromosomal region (270 kb) of S. pombe indicating enrichment of several specific genes, those discussed in the main text are labelled. Genes and genomic features are shown in black below. B ) Zoomed-in view of Upf1 enrichment over the entire pma1 gene (highlighted in dark blue) - 5’UTR (in grey), CDS (in purple) and 3’UTR (in grey) of pma1 are shown in the bottom row schematic. C ) Top panel-diagram of the pma1 gene (cDNA region in grey) and the positions of amplicons used for the PCR-ChIP assay are indicated by the dotted lines above (numbers correspond to the position of the primers relative to start codon). Bottom panel-polyacrylamide gels showing radiolabelled PCR products produced by the pma1 -specific primer pairs (top bands) and by the intergenic region specific primer (bottom bands, labelled Int.); using input DNA before ChIP (left panel) and using ChIP-enriched DNA from asynchronous culture without (middle panel) and with RNase pre-treatment of the chromatin (right panel). The relative enrichment of pma1 DNA relative to intergenic sequence is expressed as a ratio of the intensity of the same fragments produced with the input DNA. D ) PCR analysis as in C using input DNA before ChIP (left panel) and using ChIP-enriched DNA from S-phase culture without the RNase treatment (right panel). E ) Pie-charts showing the proportion of different classes of gene associated to Upf1 in asynchronous and S-phase culture of S. pombe. F ) ChIP-qPCR of Upf1-Flag enrichment over pma1 in three independent asynchronous cultures.

    Techniques Used: GWAS, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Produced, Sequencing, Real-time Polymerase Chain Reaction

    3) Product Images from "Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics"

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.049437

    PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. A . Structures of PYO, two representative fluoroquinolones (CP = ciprofloxacin, LV = levofloxacin) and two representative aminoglycosides (GM = gentamicin, TM = tobramycin). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not 11 . 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). PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI- opmD systems (right; n = 3). For full qRT-PCR dataset, see Figs. S2, S3 and S4. C . Effect of PYO on tolerance to CP and LV in glucose minimal medium (left), and to CP in SCFM (right) (all 1 µg/mL) (n = 4). PYO itself was not toxic under the experimental conditions 8 . WT made 50-80 µM PYO as measured by absorbance of the culture supernatant at 691 nm. See Fig. S5A for experimental design. D-E . Effect of PYO on lag during outgrowth after exposure to CP. A representative field of view over different time points (D; magenta = WT::mApple, green = Δ phz ::GFP; see Movie S1) is shown together with the quantification of growth area on the agarose pads at time 0 hrs and 15 hrs (E). For these experiments, a culture of each strain tested was grown and exposed to CP (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 CP (see Methods and Fig. S5D 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 Fig. S5E. Scale bar: 20 µm. F . Tolerance of Δ phz to CP (1 µg/mL) in stationary phase in the presence of different concentrations of PYO (n = 4). G . Tolerance of Δ phz to CP (1 µg/mL) 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, F – One-way ANOVA with Tukey’s HSD multiple-comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); E, G – Welch’s unpaired t- test (* p
    Figure Legend Snippet: PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. A . Structures of PYO, two representative fluoroquinolones (CP = ciprofloxacin, LV = levofloxacin) and two representative aminoglycosides (GM = gentamicin, TM = tobramycin). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not 11 . 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). PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI- opmD systems (right; n = 3). For full qRT-PCR dataset, see Figs. S2, S3 and S4. C . Effect of PYO on tolerance to CP and LV in glucose minimal medium (left), and to CP in SCFM (right) (all 1 µg/mL) (n = 4). PYO itself was not toxic under the experimental conditions 8 . WT made 50-80 µM PYO as measured by absorbance of the culture supernatant at 691 nm. See Fig. S5A for experimental design. D-E . Effect of PYO on lag during outgrowth after exposure to CP. A representative field of view over different time points (D; magenta = WT::mApple, green = Δ phz ::GFP; see Movie S1) is shown together with the quantification of growth area on the agarose pads at time 0 hrs and 15 hrs (E). For these experiments, a culture of each strain tested was grown and exposed to CP (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 CP (see Methods and Fig. S5D 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 Fig. S5E. Scale bar: 20 µm. F . Tolerance of Δ phz to CP (1 µg/mL) in stationary phase in the presence of different concentrations of PYO (n = 4). G . Tolerance of Δ phz to CP (1 µg/mL) 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, F – One-way ANOVA with Tukey’s HSD multiple-comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); E, G – Welch’s unpaired t- test (* p

    Techniques Used: Expressing, Quantitative RT-PCR

    4) Product Images from "Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics"

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

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3001093

    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
    Figure Legend 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

    Techniques Used: Expressing, Quantitative RT-PCR, Produced

    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: 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.

    Article Title: Rapid Identification of Methylase Specificity (RIMS-seq) jointly identifies methylated motifs and generates shotgun sequencing of bacterial genomes
    Article Snippet: E. coli K12 MG1655 genomic DNA was extracted from a cell culture using the DNEasy Blood and Tissue kit (69504, Qiagen). .. All the other gDNA from the bacteria presented in were isolated using the Monarch genomic DNA purification kit (T3010S, New England Biolabs). .. Xp12 phage genomic DNA was obtained from Peter Weigele and Yian-Jiun Lee at New England Biolabs.

    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/ ).

    Article Title: Rapid Identification of Methylase Specificity (RIMS-seq) jointly identifies methylated motifs and generates shotgun sequencing of bacterial genomes
    Article Snippet: E. coli K12 MG1655 genomic DNA was extracted from a cell culture using the DNEasy Blood and Tissue kit (69504, Qiagen). .. All the other gDNA from the bacteria presented in were isolated using the Monarch genomic DNA purification kit (T3010S, New England Biolabs). .. Xp12 phage genomic DNA was obtained from Peter Weigele and Yian-Jiun Lee at New England Biolabs.

    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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  • 99
    New England Biolabs monarch pcr and dna cleanup kit
    mRNA reporter design and in-cell and in-solution workflows with in-cell polysome validation. (A) Schematic for the 3’ UTR-barcoded mRNA reporter used to screen mRNA performance in a pooled format. The constant regions and barcode, which flank a variable 3’ UTR, were instrumental for amplifying and identifying hundreds of constructs simultaneously in each of the pooled experiments that comprise PERSIST-seq. The <t>DNA</t> templates for full-length mRNAs were synthesized on the Codex platform and amplified in a pooled <t>PCR</t> using primers complementary to the constant region (T7 promoter) preceding the variable 5’ UTR, and to the ‘constant3’ region following the variable 3’ UTR. (B) Summary of the workflow to progress from the individually synthesized DNA templates to the in vitro synthesized mRNA pool of 233 different constructs. We then use the same mRNA pool to screen mRNA performance in a three-pronged set of in-cell and in-solution expression and stability analyses. (C) Quality control of the 233-mRNA pool on a 1.2% formaldehyde (FA) gel stained with ethidium bromide (EtBr) after 3 hrs of in vitro transcription (IVT). The mRNA pool was analyzed before and after capping and polyadenylation. Pooled IVT is equally efficient with the starting template DNA pool with or without PCR-amplification of the DNA template pool. The three major bands corresponding to the three CDS types are indicated. The RiboRuler High Range RNA ladder (Thermo Fisher) is loaded for reference. (D) Polysome fractionation analysis of a transfected mRNA reporter. As an example, the distribution of an mRNA with short scrambled 5’ and 3’ UTRs 6 hrs after transfection into HEK293T cells was compared to the distribution of endogenous human ActB mRNA. RNA was extracted from fractions and quantified by qPCR with a RNA spike-in for normalization. Values are plotted as mRNA normalized per fraction. (E) In-solution RNA degradation strategy of barcoded mRNAs containing CDS variants with hHBB 5’ and 3’ UTRs. The differential degradation of CDS variants depends on their individual CDS structures. mRNA pools are degraded in solution by nucleophilic attack (red circle). After degradation, RT-PCR is performed to selectively amplify mRNAs that remain intact along their full length. Then, the barcode regions of these full-length mRNAs are PCR-amplified, adaptor-ligated, and prepared for Illumina sequencing.
    Monarch Pcr And Dna Cleanup 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
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    Average 99 stars, based on 1 article reviews
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    86
    New England Biolabs monarch rna cleanup kit
    5’-tRF Cys promotes Nucleolin binding to its target transcripts to enhance their stability. A. Quantification of rRNA levels upon inhibition of 5’-tRF Cys by RT-qPCR. B. Representative polysome profiles showing global translation status in 4T1 cells upon inhibition of 5’-tRF Cys . Mono, monosomes. Di, disomes. C. Percentage of Nucleolin peaks in different types of <t>RNAs.</t> RMSK, repeat masked RNAs. D. The number of Nucleolin-bound CLIP peaks in 5’, 3’ untranslated region (UTR) or coding sequencing (CDS) per 10 kb in the mouse genome. E. Cumulative distribution function (CDF) plots of log 2 FC in transcript abundance for all transcripts stratified by whether they were bound by Nucleolin (red) or not (grey). Statistical significance was determined by Kolmogorov–Smirnov (KS) test (P = 4.8e-13). F. Scatter plot comparing log 2 FC in transcript abundance upon inhibition of 5’-tRF Cys with two distinct 5’-tRF Cys antisense LNAs. Statistically significantly changed genes are marked in red. The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. G. Scatter plot comparing log 2 FC in protein abundance and log 2 FC in transcript abundance between 5’-tRF Cys suppressed and control cells for all transcripts stratified by whether their Nucleolin binding is enhanced by 5’-tRF Cys (red) or not (grey). The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. H, I. Representative western blot images of 5’-tRF Cys targets upon suppression of 5’-tRF Cys (H) or depletion of Nucleolin (I). J. Genome browser view of the aligned Nucleolin (Ncl)-CLIP tags (orange), <t>RNA-Seq</t> reads (red) and Ribo-Seq reads (green) within the 5’ UTR of Pafah1b1. The Y axis represents reads per million (RPM). TSS, transcription start site. K. Quantification by dual luciferase assays of the luminescence signals of reporters containing 5’ UTRs from 5’-tRF Cys targets relative to that from the control GAPDH. Statistical significance in A and K was determined by one-tail t-tests with Welch’s correction. ns, not significant. ***, p
    Monarch Rna Cleanup Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch rna cleanup kit/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
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    monarch rna cleanup kit - by Bioz Stars, 2021-05
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    99
    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
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    mRNA reporter design and in-cell and in-solution workflows with in-cell polysome validation. (A) Schematic for the 3’ UTR-barcoded mRNA reporter used to screen mRNA performance in a pooled format. The constant regions and barcode, which flank a variable 3’ UTR, were instrumental for amplifying and identifying hundreds of constructs simultaneously in each of the pooled experiments that comprise PERSIST-seq. The DNA templates for full-length mRNAs were synthesized on the Codex platform and amplified in a pooled PCR using primers complementary to the constant region (T7 promoter) preceding the variable 5’ UTR, and to the ‘constant3’ region following the variable 3’ UTR. (B) Summary of the workflow to progress from the individually synthesized DNA templates to the in vitro synthesized mRNA pool of 233 different constructs. We then use the same mRNA pool to screen mRNA performance in a three-pronged set of in-cell and in-solution expression and stability analyses. (C) Quality control of the 233-mRNA pool on a 1.2% formaldehyde (FA) gel stained with ethidium bromide (EtBr) after 3 hrs of in vitro transcription (IVT). The mRNA pool was analyzed before and after capping and polyadenylation. Pooled IVT is equally efficient with the starting template DNA pool with or without PCR-amplification of the DNA template pool. The three major bands corresponding to the three CDS types are indicated. The RiboRuler High Range RNA ladder (Thermo Fisher) is loaded for reference. (D) Polysome fractionation analysis of a transfected mRNA reporter. As an example, the distribution of an mRNA with short scrambled 5’ and 3’ UTRs 6 hrs after transfection into HEK293T cells was compared to the distribution of endogenous human ActB mRNA. RNA was extracted from fractions and quantified by qPCR with a RNA spike-in for normalization. Values are plotted as mRNA normalized per fraction. (E) In-solution RNA degradation strategy of barcoded mRNAs containing CDS variants with hHBB 5’ and 3’ UTRs. The differential degradation of CDS variants depends on their individual CDS structures. mRNA pools are degraded in solution by nucleophilic attack (red circle). After degradation, RT-PCR is performed to selectively amplify mRNAs that remain intact along their full length. Then, the barcode regions of these full-length mRNAs are PCR-amplified, adaptor-ligated, and prepared for Illumina sequencing.

    Journal: bioRxiv

    Article Title: Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

    doi: 10.1101/2021.03.29.437587

    Figure Lengend Snippet: mRNA reporter design and in-cell and in-solution workflows with in-cell polysome validation. (A) Schematic for the 3’ UTR-barcoded mRNA reporter used to screen mRNA performance in a pooled format. The constant regions and barcode, which flank a variable 3’ UTR, were instrumental for amplifying and identifying hundreds of constructs simultaneously in each of the pooled experiments that comprise PERSIST-seq. The DNA templates for full-length mRNAs were synthesized on the Codex platform and amplified in a pooled PCR using primers complementary to the constant region (T7 promoter) preceding the variable 5’ UTR, and to the ‘constant3’ region following the variable 3’ UTR. (B) Summary of the workflow to progress from the individually synthesized DNA templates to the in vitro synthesized mRNA pool of 233 different constructs. We then use the same mRNA pool to screen mRNA performance in a three-pronged set of in-cell and in-solution expression and stability analyses. (C) Quality control of the 233-mRNA pool on a 1.2% formaldehyde (FA) gel stained with ethidium bromide (EtBr) after 3 hrs of in vitro transcription (IVT). The mRNA pool was analyzed before and after capping and polyadenylation. Pooled IVT is equally efficient with the starting template DNA pool with or without PCR-amplification of the DNA template pool. The three major bands corresponding to the three CDS types are indicated. The RiboRuler High Range RNA ladder (Thermo Fisher) is loaded for reference. (D) Polysome fractionation analysis of a transfected mRNA reporter. As an example, the distribution of an mRNA with short scrambled 5’ and 3’ UTRs 6 hrs after transfection into HEK293T cells was compared to the distribution of endogenous human ActB mRNA. RNA was extracted from fractions and quantified by qPCR with a RNA spike-in for normalization. Values are plotted as mRNA normalized per fraction. (E) In-solution RNA degradation strategy of barcoded mRNAs containing CDS variants with hHBB 5’ and 3’ UTRs. The differential degradation of CDS variants depends on their individual CDS structures. mRNA pools are degraded in solution by nucleophilic attack (red circle). After degradation, RT-PCR is performed to selectively amplify mRNAs that remain intact along their full length. Then, the barcode regions of these full-length mRNAs are PCR-amplified, adaptor-ligated, and prepared for Illumina sequencing.

    Article Snippet: PCR reactions were purified with Monarch PCR & DNA Cleanup Kit (NEB, T1030L).

    Techniques: Construct, Synthesized, Amplification, Polymerase Chain Reaction, In Vitro, Expressing, Staining, Fractionation, Transfection, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Sequencing

    ( A ) Schematics of the construction of the recombinant plasmid. Short (TTAGGG) 5 (blue fragment) and (CCCTAA) 5 (red fragment) DNAs were extended by EM-PCR and then inserted into the T-Vector. The recombinant plasmid that contains double-stranded telomeric DNA can be obtained after overnight transfection and incubation. ( B ) Agarose gel analysis of the extension products produced from (TTAGGG) 5 and (CCCTAA) 5 during EM-PCR. Lane 1: 4 cycles; lane 2: 7 cycles; lane 3: 10 cycles; lane 4: 14 cycles; lane 5: 17 cycles; and lane 6: 20 cycles. ( C ) The T-Vector and the recombinant plasmid were double-digested by BspQI and BstAPI restriction endonucleases. The products were analyzed by agarose gel as shown in lane 1 (T-Vector) and lane 2 (recombinant, pUC18-T2AG3), respectively. ( D ) AFM imaging of telomeric-sequence-containing DNA (∼1200 bp) on mica.

    Journal: Nucleic Acids Research

    Article Title: Dynamic topology of double-stranded telomeric DNA studied by single-molecule manipulation in vitro

    doi: 10.1093/nar/gkaa479

    Figure Lengend Snippet: ( A ) Schematics of the construction of the recombinant plasmid. Short (TTAGGG) 5 (blue fragment) and (CCCTAA) 5 (red fragment) DNAs were extended by EM-PCR and then inserted into the T-Vector. The recombinant plasmid that contains double-stranded telomeric DNA can be obtained after overnight transfection and incubation. ( B ) Agarose gel analysis of the extension products produced from (TTAGGG) 5 and (CCCTAA) 5 during EM-PCR. Lane 1: 4 cycles; lane 2: 7 cycles; lane 3: 10 cycles; lane 4: 14 cycles; lane 5: 17 cycles; and lane 6: 20 cycles. ( C ) The T-Vector and the recombinant plasmid were double-digested by BspQI and BstAPI restriction endonucleases. The products were analyzed by agarose gel as shown in lane 1 (T-Vector) and lane 2 (recombinant, pUC18-T2AG3), respectively. ( D ) AFM imaging of telomeric-sequence-containing DNA (∼1200 bp) on mica.

    Article Snippet: We designed the primers such that the PCR products contained the restriction sites near the end of the strands, allowing us to remove the digested useless short DNA fragments via the PCR purification kit instead of gel purification.

    Techniques: Recombinant, Plasmid Preparation, Polymerase Chain Reaction, Transfection, Incubation, Agarose Gel Electrophoresis, Produced, Imaging, Sequencing

    5’-tRF Cys promotes Nucleolin binding to its target transcripts to enhance their stability. A. Quantification of rRNA levels upon inhibition of 5’-tRF Cys by RT-qPCR. B. Representative polysome profiles showing global translation status in 4T1 cells upon inhibition of 5’-tRF Cys . Mono, monosomes. Di, disomes. C. Percentage of Nucleolin peaks in different types of RNAs. RMSK, repeat masked RNAs. D. The number of Nucleolin-bound CLIP peaks in 5’, 3’ untranslated region (UTR) or coding sequencing (CDS) per 10 kb in the mouse genome. E. Cumulative distribution function (CDF) plots of log 2 FC in transcript abundance for all transcripts stratified by whether they were bound by Nucleolin (red) or not (grey). Statistical significance was determined by Kolmogorov–Smirnov (KS) test (P = 4.8e-13). F. Scatter plot comparing log 2 FC in transcript abundance upon inhibition of 5’-tRF Cys with two distinct 5’-tRF Cys antisense LNAs. Statistically significantly changed genes are marked in red. The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. G. Scatter plot comparing log 2 FC in protein abundance and log 2 FC in transcript abundance between 5’-tRF Cys suppressed and control cells for all transcripts stratified by whether their Nucleolin binding is enhanced by 5’-tRF Cys (red) or not (grey). The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. H, I. Representative western blot images of 5’-tRF Cys targets upon suppression of 5’-tRF Cys (H) or depletion of Nucleolin (I). J. Genome browser view of the aligned Nucleolin (Ncl)-CLIP tags (orange), RNA-Seq reads (red) and Ribo-Seq reads (green) within the 5’ UTR of Pafah1b1. The Y axis represents reads per million (RPM). TSS, transcription start site. K. Quantification by dual luciferase assays of the luminescence signals of reporters containing 5’ UTRs from 5’-tRF Cys targets relative to that from the control GAPDH. Statistical significance in A and K was determined by one-tail t-tests with Welch’s correction. ns, not significant. ***, p

    Journal: bioRxiv

    Article Title: A pro-metastatic tRNA fragment drives Nucleolin oligomerization and stabilization of bound metabolic mRNAs

    doi: 10.1101/2021.04.26.441477

    Figure Lengend Snippet: 5’-tRF Cys promotes Nucleolin binding to its target transcripts to enhance their stability. A. Quantification of rRNA levels upon inhibition of 5’-tRF Cys by RT-qPCR. B. Representative polysome profiles showing global translation status in 4T1 cells upon inhibition of 5’-tRF Cys . Mono, monosomes. Di, disomes. C. Percentage of Nucleolin peaks in different types of RNAs. RMSK, repeat masked RNAs. D. The number of Nucleolin-bound CLIP peaks in 5’, 3’ untranslated region (UTR) or coding sequencing (CDS) per 10 kb in the mouse genome. E. Cumulative distribution function (CDF) plots of log 2 FC in transcript abundance for all transcripts stratified by whether they were bound by Nucleolin (red) or not (grey). Statistical significance was determined by Kolmogorov–Smirnov (KS) test (P = 4.8e-13). F. Scatter plot comparing log 2 FC in transcript abundance upon inhibition of 5’-tRF Cys with two distinct 5’-tRF Cys antisense LNAs. Statistically significantly changed genes are marked in red. The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. G. Scatter plot comparing log 2 FC in protein abundance and log 2 FC in transcript abundance between 5’-tRF Cys suppressed and control cells for all transcripts stratified by whether their Nucleolin binding is enhanced by 5’-tRF Cys (red) or not (grey). The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. H, I. Representative western blot images of 5’-tRF Cys targets upon suppression of 5’-tRF Cys (H) or depletion of Nucleolin (I). J. Genome browser view of the aligned Nucleolin (Ncl)-CLIP tags (orange), RNA-Seq reads (red) and Ribo-Seq reads (green) within the 5’ UTR of Pafah1b1. The Y axis represents reads per million (RPM). TSS, transcription start site. K. Quantification by dual luciferase assays of the luminescence signals of reporters containing 5’ UTRs from 5’-tRF Cys targets relative to that from the control GAPDH. Statistical significance in A and K was determined by one-tail t-tests with Welch’s correction. ns, not significant. ***, p

    Article Snippet: After purified with Monarch RNA Cleanup Kit (NEB Biolabs), RNAs were polyadenylated with E. coli poly(A) polymerase (NEB Biolabs), and capped and 2’-O-methylated with Vaccinia Capping System (NEB Biolabs).

    Techniques: Binding Assay, Inhibition, Quantitative RT-PCR, Cross-linking Immunoprecipitation, Sequencing, Western Blot, RNA Sequencing Assay, Luciferase

    5’-tRF Cys promotes complex D assembly and Nucleolin oligomerization. A, B. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (A) or 5’-tRF Cys (B) using increasing amounts of Nucleolin protein. C. Quantification of complex D assembly as a function of Nucleolin concentration using purified Nucleolin protein. Bmax, specific maximum binding. h, Hill coefficient. Kd, equilibrium dissociation constant. D. Representative images of western blots of Nucleolin from Nucleolin IP that was pre-treated with different dilutions of micrococcal nuclease to remove endogenous RNAs before complexes were assembled at 30 °C and crosslinked with ethylene glycol bis (succinimidyl succinate). The number of blue dots represent the inferred number of Nucleolin monomers based on the molecular weight. E, F. Kinetics of Nucleolin complexes assembled from Pafah1b1 (E) or 5’-tRF Cys (F) using Nucleolin IP. See also Figure 5F . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. G, H. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (G) or 5’-tRF Cys (H) using increasing amount of Nucleolin IP. See also Figure 5G . I. Native gel analysis of Nucleolin complexes assembled using Nucleolin IP from Mthfd1l alone, or together with a wild-type (WT) or Nucleolin binding deficient (MUT) 5’-tRF Cys . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. J. Representative western blot of Nucleolin using Nucleolin IP incubated with or without Pafah1b1, or with both Pafah1b1 and 5’-tRF Cy at 30 °C before crosslinking with EGS. See also Figure 5I . K. Top, quantification of the protection provided by different forms of Nucleolin from degradation by a prototypical 5’- > 3’ exonuclease Terminator after conducting the assembly assay at 4 °C or 30 °C to form monomeric Nucleolin (complex A) or oligomeric Nucleolin (complex D) respectively. Bottom, representative image of denaturing PAGE analysis of the exonuclease degradation products.

    Journal: bioRxiv

    Article Title: A pro-metastatic tRNA fragment drives Nucleolin oligomerization and stabilization of bound metabolic mRNAs

    doi: 10.1101/2021.04.26.441477

    Figure Lengend Snippet: 5’-tRF Cys promotes complex D assembly and Nucleolin oligomerization. A, B. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (A) or 5’-tRF Cys (B) using increasing amounts of Nucleolin protein. C. Quantification of complex D assembly as a function of Nucleolin concentration using purified Nucleolin protein. Bmax, specific maximum binding. h, Hill coefficient. Kd, equilibrium dissociation constant. D. Representative images of western blots of Nucleolin from Nucleolin IP that was pre-treated with different dilutions of micrococcal nuclease to remove endogenous RNAs before complexes were assembled at 30 °C and crosslinked with ethylene glycol bis (succinimidyl succinate). The number of blue dots represent the inferred number of Nucleolin monomers based on the molecular weight. E, F. Kinetics of Nucleolin complexes assembled from Pafah1b1 (E) or 5’-tRF Cys (F) using Nucleolin IP. See also Figure 5F . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. G, H. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (G) or 5’-tRF Cys (H) using increasing amount of Nucleolin IP. See also Figure 5G . I. Native gel analysis of Nucleolin complexes assembled using Nucleolin IP from Mthfd1l alone, or together with a wild-type (WT) or Nucleolin binding deficient (MUT) 5’-tRF Cys . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. J. Representative western blot of Nucleolin using Nucleolin IP incubated with or without Pafah1b1, or with both Pafah1b1 and 5’-tRF Cy at 30 °C before crosslinking with EGS. See also Figure 5I . K. Top, quantification of the protection provided by different forms of Nucleolin from degradation by a prototypical 5’- > 3’ exonuclease Terminator after conducting the assembly assay at 4 °C or 30 °C to form monomeric Nucleolin (complex A) or oligomeric Nucleolin (complex D) respectively. Bottom, representative image of denaturing PAGE analysis of the exonuclease degradation products.

    Article Snippet: After purified with Monarch RNA Cleanup Kit (NEB Biolabs), RNAs were polyadenylated with E. coli poly(A) polymerase (NEB Biolabs), and capped and 2’-O-methylated with Vaccinia Capping System (NEB Biolabs).

    Techniques: Concentration Assay, Purification, Binding Assay, Western Blot, Molecular Weight, Incubation, Polyacrylamide Gel Electrophoresis

    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