l media with kanamycin  (New England Biolabs)


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    • 96
    Name:
    Monarch Plasmid Miniprep Kit
    Description:

    Catalog Number:
    T1010
    Price:
    361
    Category:
    Other Kits
    Applications:
    DNA Manipulation
    Size:
    250 preps
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    Structured Review

    New England Biolabs l media with kanamycin
    Monarch Plasmid Miniprep Kit

    https://www.bioz.com/result/l media with kanamycin/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    l media with kanamycin - by Bioz Stars, 2021-07
    96/100 stars

    Images

    1) Product Images from "Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant"

    Article Title: Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-89029-2

    CaldoCas9 based genome editing of T. thermophilus at 65 °C. ( A ) pTTCC_crtI transformants streaked out on fresh media and grown overnight to reveal pigmentation difference in the transformants and WT cells. ( B ) Agarose gel electrophoresis of products from colony PCR amplification of the genomic locus containing crtI in T. thermophilus pTTCC_crtI transformants. Expected band size of the KO allele was 1351 bp and of WT allele 2891 bp. ( C ) pTTCC_purA transformants streaked out on minimal media with and without adenine supplementation (+ Ade − Ade, respectively) to reveal the adenine auxotrophic phenotype. ( D ) Agarose gel electrophoresis of products from colony PCR amplification of genomic locus containing purA in T. thermophilus pTTCC_purA transformants. Expected band size of the KO allele was 1094 bp and the WT allele 2320 bp. ( E ) After growth of pTTCC_purA and pTTCC_crtI transformants in media without antibiotic selection, individual colonies were streaked out on media with and without kanamycin (+ Kan, − Kan, respectively) to reveal the presence or absence of the plasmid. The agarose gel images ( B , D ) have been digitally manipulated for clarity. The original images are provided in supplementary file 5 .
    Figure Legend Snippet: CaldoCas9 based genome editing of T. thermophilus at 65 °C. ( A ) pTTCC_crtI transformants streaked out on fresh media and grown overnight to reveal pigmentation difference in the transformants and WT cells. ( B ) Agarose gel electrophoresis of products from colony PCR amplification of the genomic locus containing crtI in T. thermophilus pTTCC_crtI transformants. Expected band size of the KO allele was 1351 bp and of WT allele 2891 bp. ( C ) pTTCC_purA transformants streaked out on minimal media with and without adenine supplementation (+ Ade − Ade, respectively) to reveal the adenine auxotrophic phenotype. ( D ) Agarose gel electrophoresis of products from colony PCR amplification of genomic locus containing purA in T. thermophilus pTTCC_purA transformants. Expected band size of the KO allele was 1094 bp and the WT allele 2320 bp. ( E ) After growth of pTTCC_purA and pTTCC_crtI transformants in media without antibiotic selection, individual colonies were streaked out on media with and without kanamycin (+ Kan, − Kan, respectively) to reveal the presence or absence of the plasmid. The agarose gel images ( B , D ) have been digitally manipulated for clarity. The original images are provided in supplementary file 5 .

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Selection, Plasmid Preparation

    2) Product Images from "Filter paper-based spin column method for cost-efficient DNA or RNA purification"

    Article Title: Filter paper-based spin column method for cost-efficient DNA or RNA purification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0203011

    The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.
    Figure Legend Snippet: The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.

    Techniques Used: Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Gel Extraction, Plasmid Preparation

    Related Articles

    Plasmid Preparation:

    Article Title: An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola
    Article Snippet: Vectors listed in Table were assembled by homologous recombination , in Saccharomyces cerevisiae FY843 or using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB). .. Plasmid DNA from Escherichia coli was extracted with the Monarch Plasmid Miniprep Kit (NEB) or the GeneJET Plasmid Midiprep Kit (Thermo Scientific), and plasmid DNA from S. cerevisiae using the Easy Yeast Plasmid Isolation Kit (Takara Clontech). .. Sequencing of PCR fragments and plasmids was accomplished with the Mix2Seq Kit at Eurofins Genomics.

    Article Title: Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)
    Article Snippet: .. In most cases, magnetic carriers with immobilized affinity ligands or prepared from biopolymers, synthetic polymers, porous glass or magnetic particles based on inorganic magnetic materials, showing affinity to the target nucleic acids are used for the isolation process., Some of the commonly-used commercial affinity purification kits include the QIAprep Spin Miniprep Kit (QIAGEN), the Monarch® Plasmid Miniprep Kit (NEB), and the Wizard® Plus SV Minipreps DNA Purification Systems (Promega, Madison, WI). .. In this regard, magnetic particulate materials such as beads are more preferable to be a support for solid phase pDNA isolation due to their larger binding capacity.

    Article Title: Efficient Secretion and Recombinant Production of a Lactobacillal α-amylase in Lactiplantibacillus plantarum WCFS1: Analysis and Comparison of the Secretion Using Different Signal Peptides
    Article Snippet: .. DNA Manipulation and Transformation Plasmids were isolated from E. coli NEB5α using the Monarch plasmid miniprep kit (New England Biolabs). .. PCR products and the digested fragments were purified using the Monarch DNA Gel extraction kit (New England Biolabs) and the DNA concentration was estimated by Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, United States).

    Article Title: Enterococcus faecalis Manganese Exporter MntE Alleviates Manganese Toxicity and Is Required for Mouse Gastrointestinal Colonization
    Article Snippet: The nucleotide sequence of mntE was obtained from the E. faecalis OG1RF genome via BioCyc ( ). .. The Wizard genomic DNA purification kit (Promega Corp., Madison, WI) was used for isolation of bacterial genomic DNA (gDNA), and the Monarch plasmid miniprep kit (New England BioLabs, Ipswich, MA) was used for purification of plasmids for gene expression and construction of the complement mutant. .. The Monarch DNA gel extraction kit (New England BioLabs, Ipswich, MA) was used to isolate PCR products during PCR.

    Article Title: Filter paper-based spin column method for cost-efficient DNA or RNA purification
    Article Snippet: Recharged spin columns can be easily prepared based on a preferred commercial spin column with a flat bottom and a net structure supporting the binding material, such as those Wizard SV minicolumns from Promega (Madison, WI) ( , ). .. Alternatively, spin columns with a conical (V-shape) bottom and a drip opening, such as a miniprep column from Qiagen , and a recent version adopted in NEB Monarch plasmid miniprep kit, can be recharged by reloading filter paper discs with a diameter of 5/16 inch (~8 mm) ( ). ..

    Article Title: Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)
    Article Snippet: .. pDNA isolation using commercial DNA affinity purification kits Two commercial plasmid DNA purification kits, the QIAGEN Plasmid Mini Kit (QIAGEN, Germantown, MD) and the Monarch Plasmid Miniprep Kit (NEB, New England Biolabs, Ipswich, MA), were used for pDNA isolation. .. 2 ml plasmid-containing bacterial cells were cultivated in LB medium containing proper antibiotic overnight.

    Article Title: A mitochondrial manganese superoxide dismutase involved in innate immunity is essential for the survival of Chlamys farreri.
    Article Snippet: Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system.. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri. .. Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system.. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri. .. Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system.. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri.

    Isolation:

    Article Title: An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola
    Article Snippet: Vectors listed in Table were assembled by homologous recombination , in Saccharomyces cerevisiae FY843 or using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB). .. Plasmid DNA from Escherichia coli was extracted with the Monarch Plasmid Miniprep Kit (NEB) or the GeneJET Plasmid Midiprep Kit (Thermo Scientific), and plasmid DNA from S. cerevisiae using the Easy Yeast Plasmid Isolation Kit (Takara Clontech). .. Sequencing of PCR fragments and plasmids was accomplished with the Mix2Seq Kit at Eurofins Genomics.

    Article Title: Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)
    Article Snippet: .. In most cases, magnetic carriers with immobilized affinity ligands or prepared from biopolymers, synthetic polymers, porous glass or magnetic particles based on inorganic magnetic materials, showing affinity to the target nucleic acids are used for the isolation process., Some of the commonly-used commercial affinity purification kits include the QIAprep Spin Miniprep Kit (QIAGEN), the Monarch® Plasmid Miniprep Kit (NEB), and the Wizard® Plus SV Minipreps DNA Purification Systems (Promega, Madison, WI). .. In this regard, magnetic particulate materials such as beads are more preferable to be a support for solid phase pDNA isolation due to their larger binding capacity.

    Article Title: Efficient Secretion and Recombinant Production of a Lactobacillal α-amylase in Lactiplantibacillus plantarum WCFS1: Analysis and Comparison of the Secretion Using Different Signal Peptides
    Article Snippet: .. DNA Manipulation and Transformation Plasmids were isolated from E. coli NEB5α using the Monarch plasmid miniprep kit (New England Biolabs). .. PCR products and the digested fragments were purified using the Monarch DNA Gel extraction kit (New England Biolabs) and the DNA concentration was estimated by Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, United States).

    Article Title: Enterococcus faecalis Manganese Exporter MntE Alleviates Manganese Toxicity and Is Required for Mouse Gastrointestinal Colonization
    Article Snippet: The nucleotide sequence of mntE was obtained from the E. faecalis OG1RF genome via BioCyc ( ). .. The Wizard genomic DNA purification kit (Promega Corp., Madison, WI) was used for isolation of bacterial genomic DNA (gDNA), and the Monarch plasmid miniprep kit (New England BioLabs, Ipswich, MA) was used for purification of plasmids for gene expression and construction of the complement mutant. .. The Monarch DNA gel extraction kit (New England BioLabs, Ipswich, MA) was used to isolate PCR products during PCR.

    Article Title: Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)
    Article Snippet: .. pDNA isolation using commercial DNA affinity purification kits Two commercial plasmid DNA purification kits, the QIAGEN Plasmid Mini Kit (QIAGEN, Germantown, MD) and the Monarch Plasmid Miniprep Kit (NEB, New England Biolabs, Ipswich, MA), were used for pDNA isolation. .. 2 ml plasmid-containing bacterial cells were cultivated in LB medium containing proper antibiotic overnight.

    Article Title: A mitochondrial manganese superoxide dismutase involved in innate immunity is essential for the survival of Chlamys farreri.
    Article Snippet: Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system.. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri. .. Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system.. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri. .. Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system.. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri.

    Affinity Purification:

    Article Title: Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)
    Article Snippet: .. In most cases, magnetic carriers with immobilized affinity ligands or prepared from biopolymers, synthetic polymers, porous glass or magnetic particles based on inorganic magnetic materials, showing affinity to the target nucleic acids are used for the isolation process., Some of the commonly-used commercial affinity purification kits include the QIAprep Spin Miniprep Kit (QIAGEN), the Monarch® Plasmid Miniprep Kit (NEB), and the Wizard® Plus SV Minipreps DNA Purification Systems (Promega, Madison, WI). .. In this regard, magnetic particulate materials such as beads are more preferable to be a support for solid phase pDNA isolation due to their larger binding capacity.

    Article Title: Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)
    Article Snippet: .. pDNA isolation using commercial DNA affinity purification kits Two commercial plasmid DNA purification kits, the QIAGEN Plasmid Mini Kit (QIAGEN, Germantown, MD) and the Monarch Plasmid Miniprep Kit (NEB, New England Biolabs, Ipswich, MA), were used for pDNA isolation. .. 2 ml plasmid-containing bacterial cells were cultivated in LB medium containing proper antibiotic overnight.

    DNA Purification:

    Article Title: Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)
    Article Snippet: .. In most cases, magnetic carriers with immobilized affinity ligands or prepared from biopolymers, synthetic polymers, porous glass or magnetic particles based on inorganic magnetic materials, showing affinity to the target nucleic acids are used for the isolation process., Some of the commonly-used commercial affinity purification kits include the QIAprep Spin Miniprep Kit (QIAGEN), the Monarch® Plasmid Miniprep Kit (NEB), and the Wizard® Plus SV Minipreps DNA Purification Systems (Promega, Madison, WI). .. In this regard, magnetic particulate materials such as beads are more preferable to be a support for solid phase pDNA isolation due to their larger binding capacity.

    Article Title: Enterococcus faecalis Manganese Exporter MntE Alleviates Manganese Toxicity and Is Required for Mouse Gastrointestinal Colonization
    Article Snippet: The nucleotide sequence of mntE was obtained from the E. faecalis OG1RF genome via BioCyc ( ). .. The Wizard genomic DNA purification kit (Promega Corp., Madison, WI) was used for isolation of bacterial genomic DNA (gDNA), and the Monarch plasmid miniprep kit (New England BioLabs, Ipswich, MA) was used for purification of plasmids for gene expression and construction of the complement mutant. .. The Monarch DNA gel extraction kit (New England BioLabs, Ipswich, MA) was used to isolate PCR products during PCR.

    Article Title: Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)
    Article Snippet: .. pDNA isolation using commercial DNA affinity purification kits Two commercial plasmid DNA purification kits, the QIAGEN Plasmid Mini Kit (QIAGEN, Germantown, MD) and the Monarch Plasmid Miniprep Kit (NEB, New England Biolabs, Ipswich, MA), were used for pDNA isolation. .. 2 ml plasmid-containing bacterial cells were cultivated in LB medium containing proper antibiotic overnight.

    Transformation Assay:

    Article Title: Efficient Secretion and Recombinant Production of a Lactobacillal α-amylase in Lactiplantibacillus plantarum WCFS1: Analysis and Comparison of the Secretion Using Different Signal Peptides
    Article Snippet: .. DNA Manipulation and Transformation Plasmids were isolated from E. coli NEB5α using the Monarch plasmid miniprep kit (New England Biolabs). .. PCR products and the digested fragments were purified using the Monarch DNA Gel extraction kit (New England Biolabs) and the DNA concentration was estimated by Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, United States).

    Article Title: A mitochondrial manganese superoxide dismutase involved in innate immunity is essential for the survival of Chlamys farreri.
    Article Snippet: Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system.. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri. .. Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system.. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri. .. Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system.. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri.

    Purification:

    Article Title: Enterococcus faecalis Manganese Exporter MntE Alleviates Manganese Toxicity and Is Required for Mouse Gastrointestinal Colonization
    Article Snippet: The nucleotide sequence of mntE was obtained from the E. faecalis OG1RF genome via BioCyc ( ). .. The Wizard genomic DNA purification kit (Promega Corp., Madison, WI) was used for isolation of bacterial genomic DNA (gDNA), and the Monarch plasmid miniprep kit (New England BioLabs, Ipswich, MA) was used for purification of plasmids for gene expression and construction of the complement mutant. .. The Monarch DNA gel extraction kit (New England BioLabs, Ipswich, MA) was used to isolate PCR products during PCR.

    Expressing:

    Article Title: Enterococcus faecalis Manganese Exporter MntE Alleviates Manganese Toxicity and Is Required for Mouse Gastrointestinal Colonization
    Article Snippet: The nucleotide sequence of mntE was obtained from the E. faecalis OG1RF genome via BioCyc ( ). .. The Wizard genomic DNA purification kit (Promega Corp., Madison, WI) was used for isolation of bacterial genomic DNA (gDNA), and the Monarch plasmid miniprep kit (New England BioLabs, Ipswich, MA) was used for purification of plasmids for gene expression and construction of the complement mutant. .. The Monarch DNA gel extraction kit (New England BioLabs, Ipswich, MA) was used to isolate PCR products during PCR.

    Mutagenesis:

    Article Title: Enterococcus faecalis Manganese Exporter MntE Alleviates Manganese Toxicity and Is Required for Mouse Gastrointestinal Colonization
    Article Snippet: The nucleotide sequence of mntE was obtained from the E. faecalis OG1RF genome via BioCyc ( ). .. The Wizard genomic DNA purification kit (Promega Corp., Madison, WI) was used for isolation of bacterial genomic DNA (gDNA), and the Monarch plasmid miniprep kit (New England BioLabs, Ipswich, MA) was used for purification of plasmids for gene expression and construction of the complement mutant. .. The Monarch DNA gel extraction kit (New England BioLabs, Ipswich, MA) was used to isolate PCR products during PCR.

    Clone Assay:

    Article Title: Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant
    Article Snippet: To identify clones containing the insert, colony PCR was performed with primers pTTCC_HR_ampF and pTTCC_HR_ampR. .. A few clones were cultured overnight in liquid L media with kanamycin (30 µg/ml) and plasmids miniprepped (NEB, T1010). .. The miniprepped plasmids were restricted with BspHI (NEB, R0517) and separated on agarose gel to confirm presence of inserts.

    Cell Culture:

    Article Title: Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant
    Article Snippet: To identify clones containing the insert, colony PCR was performed with primers pTTCC_HR_ampF and pTTCC_HR_ampR. .. A few clones were cultured overnight in liquid L media with kanamycin (30 µg/ml) and plasmids miniprepped (NEB, T1010). .. The miniprepped plasmids were restricted with BspHI (NEB, R0517) and separated on agarose gel to confirm presence of inserts.

    Recombinant:

    Article Title: A mitochondrial manganese superoxide dismutase involved in innate immunity is essential for the survival of Chlamys farreri.
    Article Snippet: Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system.. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri. .. Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system.. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri. .. Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system.. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri.

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    • l media with kanamycin  (New England Biolabs)
    96
    New England Biolabs l media with kanamycin
    CaldoCas9 based genome editing of T. thermophilus at 65 °C. ( A ) pTTCC_crtI transformants streaked out on fresh <t>media</t> and grown overnight to reveal pigmentation difference in the transformants and WT cells. ( B ) Agarose gel electrophoresis of products from colony PCR amplification of the genomic locus containing crtI in T. thermophilus pTTCC_crtI transformants. Expected band size of the KO allele was 1351 bp and of WT allele 2891 bp. ( C ) pTTCC_purA transformants streaked out on minimal media <t>with</t> and without adenine supplementation (+ Ade − Ade, respectively) to reveal the adenine auxotrophic phenotype. ( D ) Agarose gel electrophoresis of products from colony PCR amplification of genomic locus containing purA in T. thermophilus pTTCC_purA transformants. Expected band size of the KO allele was 1094 bp and the WT allele 2320 bp. ( E ) After growth of pTTCC_purA and pTTCC_crtI transformants in media without antibiotic selection, individual colonies were streaked out on media with and without <t>kanamycin</t> (+ Kan, − Kan, respectively) to reveal the presence or absence of the plasmid. The agarose gel images ( B , D ) have been digitally manipulated for clarity. The original images are provided in supplementary file 5 .
    L Media With Kanamycin, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/l media with kanamycin/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    l media with kanamycin - by Bioz Stars, 2021-07
    96/100 stars
    		  Buy from Supplier
    monarch pcr and dna cleanup kit  (New England Biolabs)
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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 <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.
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    monarch gdna blood lysis buffer  (New England Biolabs)
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    SHK-1 cells were electroporated with 1.4 μM Cas9:gRNA RNP and transferred to 2 separate 96-well plates. (A) After 48h, cell survival (plate1) was calculated using CellTiter Glo 2.0. (B) genomic <t>DNA</t> (plate 2) was extracted at 7 dpt, and the target sequence amplified by PCR and editing efficiency estimated using Sanger sequencing. (C) Using 1.4 μM RNP, the editing efficiency after 7 and 14 ddpt was estimated for different electroporation settings. (D) All the sequencing data, obtained from ICE analysis of Sanger sequencing of the intergenic target region from optimisation experiments (n=55) were pooled and plotted according to edit pattern.
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    Image Search Results


    CaldoCas9 based genome editing of T. thermophilus at 65 °C. ( A ) pTTCC_crtI transformants streaked out on fresh media and grown overnight to reveal pigmentation difference in the transformants and WT cells. ( B ) Agarose gel electrophoresis of products from colony PCR amplification of the genomic locus containing crtI in T. thermophilus pTTCC_crtI transformants. Expected band size of the KO allele was 1351 bp and of WT allele 2891 bp. ( C ) pTTCC_purA transformants streaked out on minimal media with and without adenine supplementation (+ Ade − Ade, respectively) to reveal the adenine auxotrophic phenotype. ( D ) Agarose gel electrophoresis of products from colony PCR amplification of genomic locus containing purA in T. thermophilus pTTCC_purA transformants. Expected band size of the KO allele was 1094 bp and the WT allele 2320 bp. ( E ) After growth of pTTCC_purA and pTTCC_crtI transformants in media without antibiotic selection, individual colonies were streaked out on media with and without kanamycin (+ Kan, − Kan, respectively) to reveal the presence or absence of the plasmid. The agarose gel images ( B , D ) have been digitally manipulated for clarity. The original images are provided in supplementary file 5 .

    Journal: Scientific Reports

    Article Title: Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant

    doi: 10.1038/s41598-021-89029-2

    Figure Lengend Snippet: CaldoCas9 based genome editing of T. thermophilus at 65 °C. ( A ) pTTCC_crtI transformants streaked out on fresh media and grown overnight to reveal pigmentation difference in the transformants and WT cells. ( B ) Agarose gel electrophoresis of products from colony PCR amplification of the genomic locus containing crtI in T. thermophilus pTTCC_crtI transformants. Expected band size of the KO allele was 1351 bp and of WT allele 2891 bp. ( C ) pTTCC_purA transformants streaked out on minimal media with and without adenine supplementation (+ Ade − Ade, respectively) to reveal the adenine auxotrophic phenotype. ( D ) Agarose gel electrophoresis of products from colony PCR amplification of genomic locus containing purA in T. thermophilus pTTCC_purA transformants. Expected band size of the KO allele was 1094 bp and the WT allele 2320 bp. ( E ) After growth of pTTCC_purA and pTTCC_crtI transformants in media without antibiotic selection, individual colonies were streaked out on media with and without kanamycin (+ Kan, − Kan, respectively) to reveal the presence or absence of the plasmid. The agarose gel images ( B , D ) have been digitally manipulated for clarity. The original images are provided in supplementary file 5 .

    Article Snippet: A few clones were cultured overnight in liquid L media with kanamycin (30 µg/ml) and plasmids miniprepped (NEB, T1010).

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Selection, Plasmid Preparation

    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

    Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic DNA. Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing PCR products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.

    Journal: The Journal of Biological Chemistry

    Article Title: The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3

    doi: 10.1074/jbc.RA120.012518

    Figure Lengend Snippet: Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic DNA. Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing PCR products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.

    Article Snippet: The 1.2-kb PCR products were purified with Monarch PCR & DNA Cleanup Kit (New England Biolabs) and sent to BGI (Shenzhen, China) for sequencing with primer 5′-GTAGCCATGGAAACAGCACA-3′.

    Techniques: Sequencing, Mutagenesis, Clone Assay, Generated, Polymerase Chain Reaction, Immunohistochemistry

    SHK-1 cells were electroporated with 1.4 μM Cas9:gRNA RNP and transferred to 2 separate 96-well plates. (A) After 48h, cell survival (plate1) was calculated using CellTiter Glo 2.0. (B) genomic DNA (plate 2) was extracted at 7 dpt, and the target sequence amplified by PCR and editing efficiency estimated using Sanger sequencing. (C) Using 1.4 μM RNP, the editing efficiency after 7 and 14 ddpt was estimated for different electroporation settings. (D) All the sequencing data, obtained from ICE analysis of Sanger sequencing of the intergenic target region from optimisation experiments (n=55) were pooled and plotted according to edit pattern.

    Journal: bioRxiv

    Article Title: Efficient genome editing in multiple salmonid cell lines using ribonucleoprotein complexes

    doi: 10.1101/2020.04.03.022038

    Figure Lengend Snippet: SHK-1 cells were electroporated with 1.4 μM Cas9:gRNA RNP and transferred to 2 separate 96-well plates. (A) After 48h, cell survival (plate1) was calculated using CellTiter Glo 2.0. (B) genomic DNA (plate 2) was extracted at 7 dpt, and the target sequence amplified by PCR and editing efficiency estimated using Sanger sequencing. (C) Using 1.4 μM RNP, the editing efficiency after 7 and 14 ddpt was estimated for different electroporation settings. (D) All the sequencing data, obtained from ICE analysis of Sanger sequencing of the intergenic target region from optimisation experiments (n=55) were pooled and plotted according to edit pattern.

    Article Snippet: Genomic DNA (gDNA) was extracted with QuickExtract buffer (Lucigen, Middleton, USA) by adding 30 μL to a well of a 96-well plate and incubating for 5 min.

    Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Electroporation