l media with kanamycin  (New England Biolabs)


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    Monarch Plasmid Miniprep Kit
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    Monarch Plasmid Miniprep Kit 250 preps
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    T1010L
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    New England Biolabs l media with kanamycin
    Monarch Plasmid Miniprep Kit
    Monarch Plasmid Miniprep Kit 250 preps
    https://www.bioz.com/result/l media with kanamycin/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    l media with kanamycin - by Bioz Stars, 2021-06
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    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

    3) 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

    4) Product Images from "Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)"

    Article Title: Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)

    Journal: Genes & Diseases

    doi: 10.1016/j.gendis.2020.04.013

    The SSMB purification process preserves pDNA integrity. (A) The homemade plasmid phEF1-eGFP (6.9 kb) was isolated from the alkaline lysis large-scale pDNA isolation protocol and subjected to different RNA removal treatments. The red box indicates the presence of bacterial RNA, while the blue asterisk indicates the absence of bacterial RNA. (B) Colony forming efficiency. Approximately 5 μg pDNA was untreated ( a ), digested with RNase A for 60 min ( b ), or RNA depleted with Mag-Bind beads ( c ), and transformed into DH10B cells by electroporation, and 10% of the transformation mix was plated onto replicates of LB/Amp plates. Representative images from each treatment are shown. (C) The effect of residual RNA on transfection efficiency in mammalian cells. Subconfluent HEK-293 cells were seeded in 12-well cell culture plates and transfected with 10% of one standard phEF1-eGFP miniprep, either untreated, or treated with RNase A digestion or Mag-Bind SSMB depletion. Both bright field (BF) and green fluorescence (GFP) images were recorded at 48 h post transfection. Representative images are shown.
    Figure Legend Snippet: The SSMB purification process preserves pDNA integrity. (A) The homemade plasmid phEF1-eGFP (6.9 kb) was isolated from the alkaline lysis large-scale pDNA isolation protocol and subjected to different RNA removal treatments. The red box indicates the presence of bacterial RNA, while the blue asterisk indicates the absence of bacterial RNA. (B) Colony forming efficiency. Approximately 5 μg pDNA was untreated ( a ), digested with RNase A for 60 min ( b ), or RNA depleted with Mag-Bind beads ( c ), and transformed into DH10B cells by electroporation, and 10% of the transformation mix was plated onto replicates of LB/Amp plates. Representative images from each treatment are shown. (C) The effect of residual RNA on transfection efficiency in mammalian cells. Subconfluent HEK-293 cells were seeded in 12-well cell culture plates and transfected with 10% of one standard phEF1-eGFP miniprep, either untreated, or treated with RNase A digestion or Mag-Bind SSMB depletion. Both bright field (BF) and green fluorescence (GFP) images were recorded at 48 h post transfection. Representative images are shown.

    Techniques Used: Purification, Plasmid Preparation, Isolation, Alkaline Lysis, Transformation Assay, Electroporation, Transfection, Cell Culture, Fluorescence

    The complete removal of bacterial RNA in most pDNA preparations is technically challenging and significantly hampers downstream applications. (A) Incomplete removal of RNA in miniprep DNA by RNase A digestion. One tenth of one standard alkaline lysis miniprep pDNA pMOK (3.1 kb) was digested with equal amount of RNase A in triplicate. The digestion reactions were terminated at the indicated time points, and analyzed on 1% agarose gels ( a ). Representative images are shown. The red box indicates the presence of bacterial RNA. The RNA bands were quantitatively analyzed by using ImageJ software ( b ). “∗∗∗”, P
    Figure Legend Snippet: The complete removal of bacterial RNA in most pDNA preparations is technically challenging and significantly hampers downstream applications. (A) Incomplete removal of RNA in miniprep DNA by RNase A digestion. One tenth of one standard alkaline lysis miniprep pDNA pMOK (3.1 kb) was digested with equal amount of RNase A in triplicate. The digestion reactions were terminated at the indicated time points, and analyzed on 1% agarose gels ( a ). Representative images are shown. The red box indicates the presence of bacterial RNA. The RNA bands were quantitatively analyzed by using ImageJ software ( b ). “∗∗∗”, P

    Techniques Used: Alkaline Lysis, Software

    Bacterial RNA in pDNA preparations can be completely depleted by using size selection magnetic beads (SSMBs). (A) The schematic representation of the RNA depletion from pDNA process using SSMBs. The pDNA prepared from alkaline lysis protocol is mixed with the Mag-Bind SSMBs at a volume ratio of 5:2 (v/v, DNA: Beads) for 10 min at room temperature ( a ). The mixture is subjected to magnet separation ( b ) and the RNA-containing supernatant is discarded, while DNA-bound beads are washed with 70% ethanol twice ( c ). After air-dry for 60 s, the pDNA is eluted from the beads with a desired volume (20–100 μl) of ddH 2 O for any downstream use ( d ). (B) A complete removal of contaminating bacterial RNA in pDNA preps. DH10B cells transformed with pMOK ( a ) or pAdTrack ( b ) were grown overnight in 2 ml LB/Kan culture and subjected to alkaline lysis miniprep procedure. The miniprep pDNA was dissolved in 40 μl ddH 2 O, mixed with 16 μl Mag-Bind beads, and followed through the process outlined in (A) . One tenth of the eluted miniprep pDNA was analyzed on 1% agarose gels, along with the same proportions of respective input samples and the discarded supernatants. The red boxes indicate the presence of bacterial RNA, while the blue asterisks indicate the absence of bacterial RNA. Representative images are shown. (C) Quantitative assessment of the DNA recovery ( a ) and RNA removal ( b ) efficiencies of the pDNA purification approach with the SSMBs.
    Figure Legend Snippet: Bacterial RNA in pDNA preparations can be completely depleted by using size selection magnetic beads (SSMBs). (A) The schematic representation of the RNA depletion from pDNA process using SSMBs. The pDNA prepared from alkaline lysis protocol is mixed with the Mag-Bind SSMBs at a volume ratio of 5:2 (v/v, DNA: Beads) for 10 min at room temperature ( a ). The mixture is subjected to magnet separation ( b ) and the RNA-containing supernatant is discarded, while DNA-bound beads are washed with 70% ethanol twice ( c ). After air-dry for 60 s, the pDNA is eluted from the beads with a desired volume (20–100 μl) of ddH 2 O for any downstream use ( d ). (B) A complete removal of contaminating bacterial RNA in pDNA preps. DH10B cells transformed with pMOK ( a ) or pAdTrack ( b ) were grown overnight in 2 ml LB/Kan culture and subjected to alkaline lysis miniprep procedure. The miniprep pDNA was dissolved in 40 μl ddH 2 O, mixed with 16 μl Mag-Bind beads, and followed through the process outlined in (A) . One tenth of the eluted miniprep pDNA was analyzed on 1% agarose gels, along with the same proportions of respective input samples and the discarded supernatants. The red boxes indicate the presence of bacterial RNA, while the blue asterisks indicate the absence of bacterial RNA. Representative images are shown. (C) Quantitative assessment of the DNA recovery ( a ) and RNA removal ( b ) efficiencies of the pDNA purification approach with the SSMBs.

    Techniques Used: Selection, Magnetic Beads, Alkaline Lysis, Transformation Assay, Purification

    5) Product Images from "Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)"

    Article Title: Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)

    Journal: Genes & Diseases

    doi: 10.1016/j.gendis.2020.04.013

    The SSMB purification process preserves pDNA integrity. (A) The homemade plasmid phEF1-eGFP (6.9 kb) was isolated from the alkaline lysis large-scale pDNA isolation protocol and subjected to different RNA removal treatments. The red box indicates the presence of bacterial RNA, while the blue asterisk indicates the absence of bacterial RNA. (B) Colony forming efficiency. Approximately 5 μg pDNA was untreated ( a ), digested with RNase A for 60 min ( b ), or RNA depleted with Mag-Bind beads ( c ), and transformed into DH10B cells by electroporation, and 10% of the transformation mix was plated onto replicates of LB/Amp plates. Representative images from each treatment are shown. (C) The effect of residual RNA on transfection efficiency in mammalian cells. Subconfluent HEK-293 cells were seeded in 12-well cell culture plates and transfected with 10% of one standard phEF1-eGFP miniprep, either untreated, or treated with RNase A digestion or Mag-Bind SSMB depletion. Both bright field (BF) and green fluorescence (GFP) images were recorded at 48 h post transfection. Representative images are shown.
    Figure Legend Snippet: The SSMB purification process preserves pDNA integrity. (A) The homemade plasmid phEF1-eGFP (6.9 kb) was isolated from the alkaline lysis large-scale pDNA isolation protocol and subjected to different RNA removal treatments. The red box indicates the presence of bacterial RNA, while the blue asterisk indicates the absence of bacterial RNA. (B) Colony forming efficiency. Approximately 5 μg pDNA was untreated ( a ), digested with RNase A for 60 min ( b ), or RNA depleted with Mag-Bind beads ( c ), and transformed into DH10B cells by electroporation, and 10% of the transformation mix was plated onto replicates of LB/Amp plates. Representative images from each treatment are shown. (C) The effect of residual RNA on transfection efficiency in mammalian cells. Subconfluent HEK-293 cells were seeded in 12-well cell culture plates and transfected with 10% of one standard phEF1-eGFP miniprep, either untreated, or treated with RNase A digestion or Mag-Bind SSMB depletion. Both bright field (BF) and green fluorescence (GFP) images were recorded at 48 h post transfection. Representative images are shown.

    Techniques Used: Purification, Plasmid Preparation, Isolation, Alkaline Lysis, Transformation Assay, Electroporation, Transfection, Cell Culture, Fluorescence

    The complete removal of bacterial RNA in most pDNA preparations is technically challenging and significantly hampers downstream applications. (A) Incomplete removal of RNA in miniprep DNA by RNase A digestion. One tenth of one standard alkaline lysis miniprep pDNA pMOK (3.1 kb) was digested with equal amount of RNase A in triplicate. The digestion reactions were terminated at the indicated time points, and analyzed on 1% agarose gels ( a ). Representative images are shown. The red box indicates the presence of bacterial RNA. The RNA bands were quantitatively analyzed by using ImageJ software ( b ). “∗∗∗”, P
    Figure Legend Snippet: The complete removal of bacterial RNA in most pDNA preparations is technically challenging and significantly hampers downstream applications. (A) Incomplete removal of RNA in miniprep DNA by RNase A digestion. One tenth of one standard alkaline lysis miniprep pDNA pMOK (3.1 kb) was digested with equal amount of RNase A in triplicate. The digestion reactions were terminated at the indicated time points, and analyzed on 1% agarose gels ( a ). Representative images are shown. The red box indicates the presence of bacterial RNA. The RNA bands were quantitatively analyzed by using ImageJ software ( b ). “∗∗∗”, P

    Techniques Used: Alkaline Lysis, Software

    Bacterial RNA in pDNA preparations can be completely depleted by using size selection magnetic beads (SSMBs). (A) The schematic representation of the RNA depletion from pDNA process using SSMBs. The pDNA prepared from alkaline lysis protocol is mixed with the Mag-Bind SSMBs at a volume ratio of 5:2 (v/v, DNA: Beads) for 10 min at room temperature ( a ). The mixture is subjected to magnet separation ( b ) and the RNA-containing supernatant is discarded, while DNA-bound beads are washed with 70% ethanol twice ( c ). After air-dry for 60 s, the pDNA is eluted from the beads with a desired volume (20–100 μl) of ddH 2 O for any downstream use ( d ). (B) A complete removal of contaminating bacterial RNA in pDNA preps. DH10B cells transformed with pMOK ( a ) or pAdTrack ( b ) were grown overnight in 2 ml LB/Kan culture and subjected to alkaline lysis miniprep procedure. The miniprep pDNA was dissolved in 40 μl ddH 2 O, mixed with 16 μl Mag-Bind beads, and followed through the process outlined in (A) . One tenth of the eluted miniprep pDNA was analyzed on 1% agarose gels, along with the same proportions of respective input samples and the discarded supernatants. The red boxes indicate the presence of bacterial RNA, while the blue asterisks indicate the absence of bacterial RNA. Representative images are shown. (C) Quantitative assessment of the DNA recovery ( a ) and RNA removal ( b ) efficiencies of the pDNA purification approach with the SSMBs.
    Figure Legend Snippet: Bacterial RNA in pDNA preparations can be completely depleted by using size selection magnetic beads (SSMBs). (A) The schematic representation of the RNA depletion from pDNA process using SSMBs. The pDNA prepared from alkaline lysis protocol is mixed with the Mag-Bind SSMBs at a volume ratio of 5:2 (v/v, DNA: Beads) for 10 min at room temperature ( a ). The mixture is subjected to magnet separation ( b ) and the RNA-containing supernatant is discarded, while DNA-bound beads are washed with 70% ethanol twice ( c ). After air-dry for 60 s, the pDNA is eluted from the beads with a desired volume (20–100 μl) of ddH 2 O for any downstream use ( d ). (B) A complete removal of contaminating bacterial RNA in pDNA preps. DH10B cells transformed with pMOK ( a ) or pAdTrack ( b ) were grown overnight in 2 ml LB/Kan culture and subjected to alkaline lysis miniprep procedure. The miniprep pDNA was dissolved in 40 μl ddH 2 O, mixed with 16 μl Mag-Bind beads, and followed through the process outlined in (A) . One tenth of the eluted miniprep pDNA was analyzed on 1% agarose gels, along with the same proportions of respective input samples and the discarded supernatants. The red boxes indicate the presence of bacterial RNA, while the blue asterisks indicate the absence of bacterial RNA. Representative images are shown. (C) Quantitative assessment of the DNA recovery ( a ) and RNA removal ( b ) efficiencies of the pDNA purification approach with the SSMBs.

    Techniques Used: Selection, Magnetic Beads, Alkaline Lysis, Transformation Assay, Purification

    Related Articles

    Isolation:

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    Plasmid Preparation:

    Article Title: Antibacterial activity and mode of action of potassium tetraborate tetrahydrate against soft-rot bacterial plant pathogens
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    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.

    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.

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    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.

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    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.

    Purification:

    Article Title: Enterococcus faecalis Manganese Exporter MntE Alleviates Manganese Toxicity and Is Required for Mouse Gastrointestinal Colonization
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    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.

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    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: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0203011

    Figure Lengend 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.

    Article Snippet: 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) ( ).

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

    The SSMB purification process preserves pDNA integrity. (A) The homemade plasmid phEF1-eGFP (6.9 kb) was isolated from the alkaline lysis large-scale pDNA isolation protocol and subjected to different RNA removal treatments. The red box indicates the presence of bacterial RNA, while the blue asterisk indicates the absence of bacterial RNA. (B) Colony forming efficiency. Approximately 5 μg pDNA was untreated ( a ), digested with RNase A for 60 min ( b ), or RNA depleted with Mag-Bind beads ( c ), and transformed into DH10B cells by electroporation, and 10% of the transformation mix was plated onto replicates of LB/Amp plates. Representative images from each treatment are shown. (C) The effect of residual RNA on transfection efficiency in mammalian cells. Subconfluent HEK-293 cells were seeded in 12-well cell culture plates and transfected with 10% of one standard phEF1-eGFP miniprep, either untreated, or treated with RNase A digestion or Mag-Bind SSMB depletion. Both bright field (BF) and green fluorescence (GFP) images were recorded at 48 h post transfection. Representative images are shown.

    Journal: Genes & Diseases

    Article Title: Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)

    doi: 10.1016/j.gendis.2020.04.013

    Figure Lengend Snippet: The SSMB purification process preserves pDNA integrity. (A) The homemade plasmid phEF1-eGFP (6.9 kb) was isolated from the alkaline lysis large-scale pDNA isolation protocol and subjected to different RNA removal treatments. The red box indicates the presence of bacterial RNA, while the blue asterisk indicates the absence of bacterial RNA. (B) Colony forming efficiency. Approximately 5 μg pDNA was untreated ( a ), digested with RNase A for 60 min ( b ), or RNA depleted with Mag-Bind beads ( c ), and transformed into DH10B cells by electroporation, and 10% of the transformation mix was plated onto replicates of LB/Amp plates. Representative images from each treatment are shown. (C) The effect of residual RNA on transfection efficiency in mammalian cells. Subconfluent HEK-293 cells were seeded in 12-well cell culture plates and transfected with 10% of one standard phEF1-eGFP miniprep, either untreated, or treated with RNase A digestion or Mag-Bind SSMB depletion. Both bright field (BF) and green fluorescence (GFP) images were recorded at 48 h post transfection. Representative images are shown.

    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).

    Techniques: Purification, Plasmid Preparation, Isolation, Alkaline Lysis, Transformation Assay, Electroporation, Transfection, Cell Culture, Fluorescence

    The complete removal of bacterial RNA in most pDNA preparations is technically challenging and significantly hampers downstream applications. (A) Incomplete removal of RNA in miniprep DNA by RNase A digestion. One tenth of one standard alkaline lysis miniprep pDNA pMOK (3.1 kb) was digested with equal amount of RNase A in triplicate. The digestion reactions were terminated at the indicated time points, and analyzed on 1% agarose gels ( a ). Representative images are shown. The red box indicates the presence of bacterial RNA. The RNA bands were quantitatively analyzed by using ImageJ software ( b ). “∗∗∗”, P

    Journal: Genes & Diseases

    Article Title: Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)

    doi: 10.1016/j.gendis.2020.04.013

    Figure Lengend Snippet: The complete removal of bacterial RNA in most pDNA preparations is technically challenging and significantly hampers downstream applications. (A) Incomplete removal of RNA in miniprep DNA by RNase A digestion. One tenth of one standard alkaline lysis miniprep pDNA pMOK (3.1 kb) was digested with equal amount of RNase A in triplicate. The digestion reactions were terminated at the indicated time points, and analyzed on 1% agarose gels ( a ). Representative images are shown. The red box indicates the presence of bacterial RNA. The RNA bands were quantitatively analyzed by using ImageJ software ( b ). “∗∗∗”, P

    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).

    Techniques: Alkaline Lysis, Software

    Bacterial RNA in pDNA preparations can be completely depleted by using size selection magnetic beads (SSMBs). (A) The schematic representation of the RNA depletion from pDNA process using SSMBs. The pDNA prepared from alkaline lysis protocol is mixed with the Mag-Bind SSMBs at a volume ratio of 5:2 (v/v, DNA: Beads) for 10 min at room temperature ( a ). The mixture is subjected to magnet separation ( b ) and the RNA-containing supernatant is discarded, while DNA-bound beads are washed with 70% ethanol twice ( c ). After air-dry for 60 s, the pDNA is eluted from the beads with a desired volume (20–100 μl) of ddH 2 O for any downstream use ( d ). (B) A complete removal of contaminating bacterial RNA in pDNA preps. DH10B cells transformed with pMOK ( a ) or pAdTrack ( b ) were grown overnight in 2 ml LB/Kan culture and subjected to alkaline lysis miniprep procedure. The miniprep pDNA was dissolved in 40 μl ddH 2 O, mixed with 16 μl Mag-Bind beads, and followed through the process outlined in (A) . One tenth of the eluted miniprep pDNA was analyzed on 1% agarose gels, along with the same proportions of respective input samples and the discarded supernatants. The red boxes indicate the presence of bacterial RNA, while the blue asterisks indicate the absence of bacterial RNA. Representative images are shown. (C) Quantitative assessment of the DNA recovery ( a ) and RNA removal ( b ) efficiencies of the pDNA purification approach with the SSMBs.

    Journal: Genes & Diseases

    Article Title: Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)

    doi: 10.1016/j.gendis.2020.04.013

    Figure Lengend Snippet: Bacterial RNA in pDNA preparations can be completely depleted by using size selection magnetic beads (SSMBs). (A) The schematic representation of the RNA depletion from pDNA process using SSMBs. The pDNA prepared from alkaline lysis protocol is mixed with the Mag-Bind SSMBs at a volume ratio of 5:2 (v/v, DNA: Beads) for 10 min at room temperature ( a ). The mixture is subjected to magnet separation ( b ) and the RNA-containing supernatant is discarded, while DNA-bound beads are washed with 70% ethanol twice ( c ). After air-dry for 60 s, the pDNA is eluted from the beads with a desired volume (20–100 μl) of ddH 2 O for any downstream use ( d ). (B) A complete removal of contaminating bacterial RNA in pDNA preps. DH10B cells transformed with pMOK ( a ) or pAdTrack ( b ) were grown overnight in 2 ml LB/Kan culture and subjected to alkaline lysis miniprep procedure. The miniprep pDNA was dissolved in 40 μl ddH 2 O, mixed with 16 μl Mag-Bind beads, and followed through the process outlined in (A) . One tenth of the eluted miniprep pDNA was analyzed on 1% agarose gels, along with the same proportions of respective input samples and the discarded supernatants. The red boxes indicate the presence of bacterial RNA, while the blue asterisks indicate the absence of bacterial RNA. Representative images are shown. (C) Quantitative assessment of the DNA recovery ( a ) and RNA removal ( b ) efficiencies of the pDNA purification approach with the 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).

    Techniques: Selection, Magnetic Beads, Alkaline Lysis, Transformation Assay, Purification