bamhi  (Thermo Fisher)


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  • 99
    Name:
    BamHI 10 U µL
    Description:
    5 G ↓G A T C C 3 3 C C T A G ↑G 5 Thermo Scientific BamHI restriction enzyme recognizes G GATCC sites and cuts best at 37°C in its own unique buffer See Reaction Conditions for Restriction Enzymes for a table of enzyme activity conditions for double digestion and heat inactivation for this and other restriction enzymes Note Also available as a FastDigest enzyme for rapid DNA digestion Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes optimized to work in one of the buffers of the Five Buffer System In addition the universal Tango buffer is provided for convenience in double digestions All of the enzymes exhibit 100 activity in the recommended buffer and reaction conditions To ensure consistent performance Thermo Scientific restriction enzyme reaction buffers contain premixed BSA which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations Features• Superior quality stringent quality control and industry leading manufacturing process• Convenient color coded Five Buffer System• Includes universal Tango buffer for double digestions• BSA premixed in reaction buffers• Wide selection of restriction endonuclease specificitiesApplications• Molecular cloning• Restriction site mapping• Genotyping• Southern blotting• Restriction fragment length polymorphism RFLP • SNPNote For methylation sensitivity refer to product specifications
    Catalog Number:
    er0051
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    Thermo Fisher bamhi
    A: pFastBac1 and pBlueScript-ORF2-NSP4 Genes is Double Digested With <t>BamHI</t> and <t>SalI</t> RESTRICTION ENZYMES A: The linearized pFastBac1 and ORF2-NSP4 respectively appeared at 4775 and 2000 bp. Lane 1: pFastBac1 is double digested with BamHI/SalI ; Lane 2: pBlueScript-ORF2-NSP4 is double digested with BamHI/SalI.B : ORF2-NSP4 gene was cloned in pFastBac1. Lane 1 - 2: pFastBac1-ORF2-NSP4; and Lane3 - 4: pFastBac1. C: the position site of ORF2-NSP4 was confirmed by single and double digestion and the length of subcloned genes after single digestion was 6775 bp while after double digestion was 4775 and 2000 bp. Lane 1 - 2: pFastBac1-ORF2-NSP4 single digestion BamHI ; Lane 3 - 4: pFastBac1-ORF2-NSP4 single digestion SalI; and Lane 5-6: pFastBac1-ORF2-NSP4 double digestion BamHI/SalI .
    5 G ↓G A T C C 3 3 C C T A G ↑G 5 Thermo Scientific BamHI restriction enzyme recognizes G GATCC sites and cuts best at 37°C in its own unique buffer See Reaction Conditions for Restriction Enzymes for a table of enzyme activity conditions for double digestion and heat inactivation for this and other restriction enzymes Note Also available as a FastDigest enzyme for rapid DNA digestion Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes optimized to work in one of the buffers of the Five Buffer System In addition the universal Tango buffer is provided for convenience in double digestions All of the enzymes exhibit 100 activity in the recommended buffer and reaction conditions To ensure consistent performance Thermo Scientific restriction enzyme reaction buffers contain premixed BSA which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations Features• Superior quality stringent quality control and industry leading manufacturing process• Convenient color coded Five Buffer System• Includes universal Tango buffer for double digestions• BSA premixed in reaction buffers• Wide selection of restriction endonuclease specificitiesApplications• Molecular cloning• Restriction site mapping• Genotyping• Southern blotting• Restriction fragment length polymorphism RFLP • SNPNote For methylation sensitivity refer to product specifications
    https://www.bioz.com/result/bamhi/product/Thermo Fisher
    Average 99 stars, based on 1212 article reviews
    Price from $9.99 to $1999.99
    bamhi - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System"

    Article Title: Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System

    Journal: Jundishapur Journal of Microbiology

    doi: 10.5812/jjm.40303

    A: pFastBac1 and pBlueScript-ORF2-NSP4 Genes is Double Digested With BamHI and SalI RESTRICTION ENZYMES A: The linearized pFastBac1 and ORF2-NSP4 respectively appeared at 4775 and 2000 bp. Lane 1: pFastBac1 is double digested with BamHI/SalI ; Lane 2: pBlueScript-ORF2-NSP4 is double digested with BamHI/SalI.B : ORF2-NSP4 gene was cloned in pFastBac1. Lane 1 - 2: pFastBac1-ORF2-NSP4; and Lane3 - 4: pFastBac1. C: the position site of ORF2-NSP4 was confirmed by single and double digestion and the length of subcloned genes after single digestion was 6775 bp while after double digestion was 4775 and 2000 bp. Lane 1 - 2: pFastBac1-ORF2-NSP4 single digestion BamHI ; Lane 3 - 4: pFastBac1-ORF2-NSP4 single digestion SalI; and Lane 5-6: pFastBac1-ORF2-NSP4 double digestion BamHI/SalI .
    Figure Legend Snippet: A: pFastBac1 and pBlueScript-ORF2-NSP4 Genes is Double Digested With BamHI and SalI RESTRICTION ENZYMES A: The linearized pFastBac1 and ORF2-NSP4 respectively appeared at 4775 and 2000 bp. Lane 1: pFastBac1 is double digested with BamHI/SalI ; Lane 2: pBlueScript-ORF2-NSP4 is double digested with BamHI/SalI.B : ORF2-NSP4 gene was cloned in pFastBac1. Lane 1 - 2: pFastBac1-ORF2-NSP4; and Lane3 - 4: pFastBac1. C: the position site of ORF2-NSP4 was confirmed by single and double digestion and the length of subcloned genes after single digestion was 6775 bp while after double digestion was 4775 and 2000 bp. Lane 1 - 2: pFastBac1-ORF2-NSP4 single digestion BamHI ; Lane 3 - 4: pFastBac1-ORF2-NSP4 single digestion SalI; and Lane 5-6: pFastBac1-ORF2-NSP4 double digestion BamHI/SalI .

    Techniques Used: Clone Assay

    2) Product Images from "Human Mre11/Human Rad50/Nbs1 and DNA Ligase III?/XRCC1 Protein Complexes Act Together in an Alternative Nonhomologous End Joining Pathway *"

    Article Title: Human Mre11/Human Rad50/Nbs1 and DNA Ligase III?/XRCC1 Protein Complexes Act Together in an Alternative Nonhomologous End Joining Pathway *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.274159

    Joining of incompatible DNA ends by MRN and DNA ligase IIIα/XRCC1: microhomology-mediated joining of DNA ends with incompatible 5′ overhangs. A , circular plasmid DNA was digested with either KpnI ( left ) and PstI ( right ) to generate noncomplementary 3′ overhangs or EcoRI ( left ) and BamHI ( right ), to generate noncomplementary 5′ overhangs. Joining of the incompatible DNA ends was detected by the PCR using the indicated primers. B , DNA substrate (0.8 pmol) with noncomplementary 3′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. C , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. D , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of either DNA ligase IIIα/XRCC1 or DNA ligase IV/XRCC4 in the absence or presence of an equimolar amount of MRN. E , sequences of the junctions generated by the joining of incompatible 5′ ends by DNA ligase IIIα/XRCC1 in combination with either hMre11 or MRN are shown. Bolded residues indicate microhomologies.
    Figure Legend Snippet: Joining of incompatible DNA ends by MRN and DNA ligase IIIα/XRCC1: microhomology-mediated joining of DNA ends with incompatible 5′ overhangs. A , circular plasmid DNA was digested with either KpnI ( left ) and PstI ( right ) to generate noncomplementary 3′ overhangs or EcoRI ( left ) and BamHI ( right ), to generate noncomplementary 5′ overhangs. Joining of the incompatible DNA ends was detected by the PCR using the indicated primers. B , DNA substrate (0.8 pmol) with noncomplementary 3′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. C , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. D , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of either DNA ligase IIIα/XRCC1 or DNA ligase IV/XRCC4 in the absence or presence of an equimolar amount of MRN. E , sequences of the junctions generated by the joining of incompatible 5′ ends by DNA ligase IIIα/XRCC1 in combination with either hMre11 or MRN are shown. Bolded residues indicate microhomologies.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Incubation, Generated

    3) Product Images from "Molecular and antigenic characterization of bovine herpesvirus type 1 (BoHV-1) strains from cattle with diverse clinical cases in Turkey"

    Article Title: Molecular and antigenic characterization of bovine herpesvirus type 1 (BoHV-1) strains from cattle with diverse clinical cases in Turkey

    Journal: Tropical Animal Health and Production

    doi: 10.1007/s11250-019-02042-6

    REA of BoHV-1 isolates and reference strain by BamHI (A), HindIII (B), and EcoRI (C). Lane (1):DNA ladder (100 bp plus and 1kbp plus, ThermoScientific), Lane (2): KY748023 (KYS-73675-Milk), Lane (3): KY748022 (TGM-IZ41-Milk), Lane (4): KY748020 (ANK-SKR-Lng), Lane (5): KY748021 (Halk-THYM-Thm), Lane (6): Cooper strain. REA pattern differences indicated by *; KY748022 (Lane3) and KY748021 (Lane 5) were BoHV-1.2a while the other viruses in the test were BoHV-1.1 strains
    Figure Legend Snippet: REA of BoHV-1 isolates and reference strain by BamHI (A), HindIII (B), and EcoRI (C). Lane (1):DNA ladder (100 bp plus and 1kbp plus, ThermoScientific), Lane (2): KY748023 (KYS-73675-Milk), Lane (3): KY748022 (TGM-IZ41-Milk), Lane (4): KY748020 (ANK-SKR-Lng), Lane (5): KY748021 (Halk-THYM-Thm), Lane (6): Cooper strain. REA pattern differences indicated by *; KY748022 (Lane3) and KY748021 (Lane 5) were BoHV-1.2a while the other viruses in the test were BoHV-1.1 strains

    Techniques Used:

    4) Product Images from "Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance"

    Article Title: Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance

    Journal: Jundishapur Journal of Microbiology

    doi: 10.5812/jjm.29384

    Confirmation of Self-Ligated pP15A, kana R by Digestion Lane 2: digestion of pP15A, kana R vector with BamHI ; lane 3, xbaI; Lane 4, double digestion with BamHI-XbaI; Lane 5, undigested pP15A, kana R vector; Lanes 1 and 6, GeneRuler DNA ladder mix (Thermo Scientific, Waltham, MA, USA).
    Figure Legend Snippet: Confirmation of Self-Ligated pP15A, kana R by Digestion Lane 2: digestion of pP15A, kana R vector with BamHI ; lane 3, xbaI; Lane 4, double digestion with BamHI-XbaI; Lane 5, undigested pP15A, kana R vector; Lanes 1 and 6, GeneRuler DNA ladder mix (Thermo Scientific, Waltham, MA, USA).

    Techniques Used: Plasmid Preparation

    5) Product Images from "Mapping and mutation of the conserved DNA polymerase interaction motif (DPIM) located in the C-terminal domain of fission yeast DNA polymerase ? subunit Cdc27"

    Article Title: Mapping and mutation of the conserved DNA polymerase interaction motif (DPIM) located in the C-terminal domain of fission yeast DNA polymerase ? subunit Cdc27

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-5-21

    Construction and analysis of cdc27-Q1 mutant yeast. A. Upper part: Schematic of the cdc27 + gene region (3.1 kb HindIII-BamHI region) showing location of oligonucleotides used for PCR amplification. (Key to oligonucleotides: A w = CDC27-Q1W-DIAG2, A m = CDC27-Q1M-DIAG2, B = CDC27-B, C = CDC27-SEQ2005, D = CDC27-H, X = CDC1-AB, and Y = CDC1-XY – see Material and methods for sequences). The boxes indicate approximate positions of cdc27 + exons. The NotI site shown is found in the cdc27-Q1 allele only. Lower part: Genomic DNA prepared from wild-type (WT) and cdc27-Q1 (Q1) strains was amplified using the primer pairs shown. The D+B PCR product from cdc27-Q1 alone can be digested with NotI (data not shown). The primer pair X and Y amplify an unrelated region of genome, and were included as a control. Molecular weight markers (kb) are shown to the right of the gel. B. Wild-type ( cdc27 + , left) and cdc27-Q1 (right) cells plated on YE medium and incubated for 3 days at 32°C. Mutation of the DPIM did not affect the efficiency of colony formation or growth rate. See text for details.
    Figure Legend Snippet: Construction and analysis of cdc27-Q1 mutant yeast. A. Upper part: Schematic of the cdc27 + gene region (3.1 kb HindIII-BamHI region) showing location of oligonucleotides used for PCR amplification. (Key to oligonucleotides: A w = CDC27-Q1W-DIAG2, A m = CDC27-Q1M-DIAG2, B = CDC27-B, C = CDC27-SEQ2005, D = CDC27-H, X = CDC1-AB, and Y = CDC1-XY – see Material and methods for sequences). The boxes indicate approximate positions of cdc27 + exons. The NotI site shown is found in the cdc27-Q1 allele only. Lower part: Genomic DNA prepared from wild-type (WT) and cdc27-Q1 (Q1) strains was amplified using the primer pairs shown. The D+B PCR product from cdc27-Q1 alone can be digested with NotI (data not shown). The primer pair X and Y amplify an unrelated region of genome, and were included as a control. Molecular weight markers (kb) are shown to the right of the gel. B. Wild-type ( cdc27 + , left) and cdc27-Q1 (right) cells plated on YE medium and incubated for 3 days at 32°C. Mutation of the DPIM did not affect the efficiency of colony formation or growth rate. See text for details.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Molecular Weight, Incubation

    6) Product Images from "Bacteriophage application for biocontrolling Shigella flexneri in contaminated foods"

    Article Title: Bacteriophage application for biocontrolling Shigella flexneri in contaminated foods

    Journal: Journal of Food Science and Technology

    doi: 10.1007/s13197-017-2964-2

    Phage vB_SflS-ISF001 genomic DNA size. Lane M: X-large DNA marker (SinaClon, Iran); lane 1: Untreated genomic DNA; lane 2: EcoRI; lane 3: EcoRV; lane 4: HindIII; lane 5: BamHI
    Figure Legend Snippet: Phage vB_SflS-ISF001 genomic DNA size. Lane M: X-large DNA marker (SinaClon, Iran); lane 1: Untreated genomic DNA; lane 2: EcoRI; lane 3: EcoRV; lane 4: HindIII; lane 5: BamHI

    Techniques Used: Marker

    7) Product Images from "Construction of a Novel DNA Vaccine Candidate Encoding an HspX-PPE44-EsxV Fusion Antigen of Mycobacterium tuberculosis"

    Article Title: Construction of a Novel DNA Vaccine Candidate Encoding an HspX-PPE44-EsxV Fusion Antigen of Mycobacterium tuberculosis

    Journal: Reports of Biochemistry & Molecular Biology

    doi:

    Colony-PCR and digestion products visualized on 1.2% agarose gels. A: Agarose gel electrophoresis of colony-PCR product. Lane 1: 1968 bp PCR product of fusion fragments, M: 100 bp-plus DNA size marker; B: BamHI and Xba XbaI digestion products of recombinant
    Figure Legend Snippet: Colony-PCR and digestion products visualized on 1.2% agarose gels. A: Agarose gel electrophoresis of colony-PCR product. Lane 1: 1968 bp PCR product of fusion fragments, M: 100 bp-plus DNA size marker; B: BamHI and Xba XbaI digestion products of recombinant

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Recombinant

    Agarose gels of recombinant vector. a: digestion of pGH/HspX-PPE44-EsxV by BamHI and XbaI . lane 1: pGH (2940 bp) and HspX-PPE4-EsxV (1968 bp); lane; M: 1kb DNA size marker. B: Ligated pcDNA3.1 (+)/hspX-ppe44-esxV. The band is Recombinant pcDNA3.1 (+)
    Figure Legend Snippet: Agarose gels of recombinant vector. a: digestion of pGH/HspX-PPE44-EsxV by BamHI and XbaI . lane 1: pGH (2940 bp) and HspX-PPE4-EsxV (1968 bp); lane; M: 1kb DNA size marker. B: Ligated pcDNA3.1 (+)/hspX-ppe44-esxV. The band is Recombinant pcDNA3.1 (+)

    Techniques Used: Recombinant, Plasmid Preparation, Marker

    Schematic map of the pcDNA3.1 (+)/HspX-PPE44-EsxV-His plasmid. The fusion segment consisting of hspX, linker, ppe44, linker, esxV sequences, and a 6-polyhistidine-tag was designed between the BamHI and XbaI restriction sites of pcDNA3.1 (+) downstream
    Figure Legend Snippet: Schematic map of the pcDNA3.1 (+)/HspX-PPE44-EsxV-His plasmid. The fusion segment consisting of hspX, linker, ppe44, linker, esxV sequences, and a 6-polyhistidine-tag was designed between the BamHI and XbaI restriction sites of pcDNA3.1 (+) downstream

    Techniques Used: Plasmid Preparation

    8) Product Images from "The DNA Methylome of the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius"

    Article Title: The DNA Methylome of the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00137

    Identification of methylated recognition sequences in S. acidocaldarius genomic DNA by digestion assays. Restriction enzymes BsuRI (A) and BamHI, DpnI, and MboI (B) were used to highlight the presence/absence of methylated 5′-GGCC-3′ and 5′-GATC-3′ palindromes, respectively, in genomic DNA of S. acidocaldarius . Two types of substrates were digested: genomic DNA (gDNA) and a whole genome amplification of the genomic DNA (WGA). WGA is identical to gDNA in terms of nucleic sequence but it does not contain epigenetic marks (see material and methods). This negative control is only used in (A) . The addition of different restriction enzymes is symbolized by a positive sign “+” while reaction mix without restriction enzyme is represented by a negative sign “–”. Molecular size markers (GeneRuler DNA Ladder, Thermo Scientific) were loaded in both gels (M). Digestion patterns were obtained in 0.8% agarose gel stained with ethidium bromide and visualized under UV light.
    Figure Legend Snippet: Identification of methylated recognition sequences in S. acidocaldarius genomic DNA by digestion assays. Restriction enzymes BsuRI (A) and BamHI, DpnI, and MboI (B) were used to highlight the presence/absence of methylated 5′-GGCC-3′ and 5′-GATC-3′ palindromes, respectively, in genomic DNA of S. acidocaldarius . Two types of substrates were digested: genomic DNA (gDNA) and a whole genome amplification of the genomic DNA (WGA). WGA is identical to gDNA in terms of nucleic sequence but it does not contain epigenetic marks (see material and methods). This negative control is only used in (A) . The addition of different restriction enzymes is symbolized by a positive sign “+” while reaction mix without restriction enzyme is represented by a negative sign “–”. Molecular size markers (GeneRuler DNA Ladder, Thermo Scientific) were loaded in both gels (M). Digestion patterns were obtained in 0.8% agarose gel stained with ethidium bromide and visualized under UV light.

    Techniques Used: Methylation, Whole Genome Amplification, Sequencing, Negative Control, Agarose Gel Electrophoresis, Staining

    9) Product Images from "Alopecia in a Viable Phospholipase C Delta 1 and Phospholipase C Delta 3 Double Mutant"

    Article Title: Alopecia in a Viable Phospholipase C Delta 1 and Phospholipase C Delta 3 Double Mutant

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039203

    Molecular analysis of the Plcd3 mNab mutation. A. Schematic representation of the genomic context of the Plcd3 mNab mutation. The Plcd3 mNab mutation is caused by the insertion of 5.4 kbp of IAP sequences in intron 2 of Plcd3 . Hybridisation with a probe (P) derived from intron 1 using primers 572 and 573 ( Table S1 ) reveals a restriction fragment length polymorphism in BamHI digested genomic DNA of wild-type (+/+), oltSH (+/−) and oltNH (−/−) mice, respectively. The wild-type allele shows a 4.1 kbp fragment and the mutant allele a fragment of 4.5 kbp. The increased length of the mutant fragment is caused by the BamHI site (B) within the inserted IAP. The Plcd3 locus is shown in reverse orientation with respect to the chromosomal DNA, with Plcd3 exon 1 to the left. The intracisternal A particle (IAP) has no env sequences and is inserted in intron 2 in reverse orientation with respect to the transcription of the Plcd3 gene. B. Location of the primers used to analyse the Plcd3 mNab mutant and the wild-type Plcd3 locus (f, forward; r, reverse). On the left, electrophoresis of amplified genomic DNA fragments that are indicative of the wild-type Plcd3 and the Plcd3 mNab allele using the primers indicated. On the right, the PCR products from genomic DNA around Plcd3 intron 2 obtained from wild-type and oltNH mice using primers f1046 and r1049 ( Table S1 ) are shown. The 5.4 kbp long fragment in the oltNH mutant contains the inserted IAP sequence. C. Northern blot analysis of mutant dorsal skin. A DIG-labelled probe derived from the 5′ region of Plcd1 hybridises to transcripts only in phenotypically wild-type animals. A DIG-labelled RNA probe derived from the 3′ region of Plcd3 hybridises to two transcripts of 3 kb and 2.6 kb, respectively (marked by arrowheads). oltSH mice express both transcripts, while oltNH mice show only the 2.6 kb transcript. Note that the wild-type mouse in lane 2 shows the mutant Plcd3 transcript and expresses Plcd1 . Hybridisation with a Gapdh -specific probe as loading control is also shown. D. Western blot analysis of mutant dorsal skin using antibody IF12–15 directed against the catalytic region of PLCD3 protein and 4H 5–9 directed against the PH domain of PLCD3. The phenotypes of the mice (Wt, oltNH , oltSH ), from which the lysates were obtained, are given on top of each lane. The 2.6 kb mutant Plcd3 transcript is translated to a truncated protein of 75 kDa, which is detected by the antibody IF12–15, but not the PH domain-specific antibody 4H 5–9 (lanes 2 and 3). This antibody also bound to an unknown protein of 110 kDa in all samples. Arrowheads indicate the wild-type protein. An immunoblot using antibody against actin is given below as a control. Note that the phenotypically wild-type in lane 2 is the same as the one in lane 2 of the Northern blot in C.
    Figure Legend Snippet: Molecular analysis of the Plcd3 mNab mutation. A. Schematic representation of the genomic context of the Plcd3 mNab mutation. The Plcd3 mNab mutation is caused by the insertion of 5.4 kbp of IAP sequences in intron 2 of Plcd3 . Hybridisation with a probe (P) derived from intron 1 using primers 572 and 573 ( Table S1 ) reveals a restriction fragment length polymorphism in BamHI digested genomic DNA of wild-type (+/+), oltSH (+/−) and oltNH (−/−) mice, respectively. The wild-type allele shows a 4.1 kbp fragment and the mutant allele a fragment of 4.5 kbp. The increased length of the mutant fragment is caused by the BamHI site (B) within the inserted IAP. The Plcd3 locus is shown in reverse orientation with respect to the chromosomal DNA, with Plcd3 exon 1 to the left. The intracisternal A particle (IAP) has no env sequences and is inserted in intron 2 in reverse orientation with respect to the transcription of the Plcd3 gene. B. Location of the primers used to analyse the Plcd3 mNab mutant and the wild-type Plcd3 locus (f, forward; r, reverse). On the left, electrophoresis of amplified genomic DNA fragments that are indicative of the wild-type Plcd3 and the Plcd3 mNab allele using the primers indicated. On the right, the PCR products from genomic DNA around Plcd3 intron 2 obtained from wild-type and oltNH mice using primers f1046 and r1049 ( Table S1 ) are shown. The 5.4 kbp long fragment in the oltNH mutant contains the inserted IAP sequence. C. Northern blot analysis of mutant dorsal skin. A DIG-labelled probe derived from the 5′ region of Plcd1 hybridises to transcripts only in phenotypically wild-type animals. A DIG-labelled RNA probe derived from the 3′ region of Plcd3 hybridises to two transcripts of 3 kb and 2.6 kb, respectively (marked by arrowheads). oltSH mice express both transcripts, while oltNH mice show only the 2.6 kb transcript. Note that the wild-type mouse in lane 2 shows the mutant Plcd3 transcript and expresses Plcd1 . Hybridisation with a Gapdh -specific probe as loading control is also shown. D. Western blot analysis of mutant dorsal skin using antibody IF12–15 directed against the catalytic region of PLCD3 protein and 4H 5–9 directed against the PH domain of PLCD3. The phenotypes of the mice (Wt, oltNH , oltSH ), from which the lysates were obtained, are given on top of each lane. The 2.6 kb mutant Plcd3 transcript is translated to a truncated protein of 75 kDa, which is detected by the antibody IF12–15, but not the PH domain-specific antibody 4H 5–9 (lanes 2 and 3). This antibody also bound to an unknown protein of 110 kDa in all samples. Arrowheads indicate the wild-type protein. An immunoblot using antibody against actin is given below as a control. Note that the phenotypically wild-type in lane 2 is the same as the one in lane 2 of the Northern blot in C.

    Techniques Used: Mutagenesis, Hybridization, Derivative Assay, Mouse Assay, Electrophoresis, Amplification, Polymerase Chain Reaction, Sequencing, Northern Blot, Western Blot

    10) Product Images from "Characterization of RNA Helicase CshA and Its Role in Protecting mRNAs and Small RNAs of Staphylococcus aureus Strain Newman"

    Article Title: Characterization of RNA Helicase CshA and Its Role in Protecting mRNAs and Small RNAs of Staphylococcus aureus Strain Newman

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01042-15

    Interaction of sarA mRNA with truncated versions of CshA. (A) Depiction of the different truncated versions of CshA. Each truncated fragment was created by PCR, cloned to pET22b with NdeI and BamHI, and expressed using E. coli BL21(pLysS). (B) SDS-PAGE (12%) analyzing cell lysates before (at an OD 600 of 0.7) and after addition of IPTG to a final concentration of 0.2 mM and grown for 3 h [indicated by (−) and (+), respectively]. (C) Northwestern blot showing labeled sarA mRNA hybridized to protein bands.
    Figure Legend Snippet: Interaction of sarA mRNA with truncated versions of CshA. (A) Depiction of the different truncated versions of CshA. Each truncated fragment was created by PCR, cloned to pET22b with NdeI and BamHI, and expressed using E. coli BL21(pLysS). (B) SDS-PAGE (12%) analyzing cell lysates before (at an OD 600 of 0.7) and after addition of IPTG to a final concentration of 0.2 mM and grown for 3 h [indicated by (−) and (+), respectively]. (C) Northwestern blot showing labeled sarA mRNA hybridized to protein bands.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, SDS Page, Concentration Assay, Labeling

    11) Product Images from "Development of a Novel PCR Restriction Analysis of the hsp65 Gene as a Rapid Method To Screen for the Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in High-Burden Countries"

    Article Title: Development of a Novel PCR Restriction Analysis of the hsp65 Gene as a Rapid Method To Screen for the Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in High-Burden Countries

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.03067-12

    NruI and BamHI digests of amplicons from the PCR conducted on serial dilutions of H37Rv in a spiked sputum sample with the HSP N3 and N4 primers. No PCR amplicon was obtained with the spiked specimen with 10 0 organisms/μl. Lane 1, undigested H37Rv amplicon. Lanes 2 and 9, 50-bp marker. Lanes 3 to 8, NruI digests of PCR amplicons from a smear-negative sputum sample spiked with 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , or 10 6 organisms/μl, respectively. Lanes 10 to 15, BamHI digests of PCR amplicons from a smear-negative sputum sample spiked with 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , or 10 6 organisms/μl, respectively.
    Figure Legend Snippet: NruI and BamHI digests of amplicons from the PCR conducted on serial dilutions of H37Rv in a spiked sputum sample with the HSP N3 and N4 primers. No PCR amplicon was obtained with the spiked specimen with 10 0 organisms/μl. Lane 1, undigested H37Rv amplicon. Lanes 2 and 9, 50-bp marker. Lanes 3 to 8, NruI digests of PCR amplicons from a smear-negative sputum sample spiked with 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , or 10 6 organisms/μl, respectively. Lanes 10 to 15, BamHI digests of PCR amplicons from a smear-negative sputum sample spiked with 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , or 10 6 organisms/μl, respectively.

    Techniques Used: Polymerase Chain Reaction, Amplification, Marker

    12) Product Images from "Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences"

    Article Title: Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences

    Journal: Genome Research

    doi: 10.1101/gr.094953.109

    Analysis of DNA fractions obtained by SRD and MND treatment of sperm nuclei. All DNA samples were resolved on 1.8% agarose gels. (Lane 1 ) A 0.4–10-kb DNA ladder. (Lane 2 ) Total (unfractionated) DNA (NF). (Lane 3 ) The soluble DNA released after extraction of sperm nuclei with 0.65 M NaCl followed by digestion with BamHI and EcoRI (SRDS). (Lane 5 ) The corresponding insoluble pellet (SRDI). (Lane 4 ) DNA released from sperm after MNase digestion (MNDS). (Lane 6 ) The corresponding insoluble pellet (MNDI).
    Figure Legend Snippet: Analysis of DNA fractions obtained by SRD and MND treatment of sperm nuclei. All DNA samples were resolved on 1.8% agarose gels. (Lane 1 ) A 0.4–10-kb DNA ladder. (Lane 2 ) Total (unfractionated) DNA (NF). (Lane 3 ) The soluble DNA released after extraction of sperm nuclei with 0.65 M NaCl followed by digestion with BamHI and EcoRI (SRDS). (Lane 5 ) The corresponding insoluble pellet (SRDI). (Lane 4 ) DNA released from sperm after MNase digestion (MNDS). (Lane 6 ) The corresponding insoluble pellet (MNDI).

    Techniques Used:

    13) Product Images from "Isolation and Characterization of vB_ArS-ArV2 – First Arthrobacter sp. Infecting Bacteriophage with Completely Sequenced Genome"

    Article Title: Isolation and Characterization of vB_ArS-ArV2 – First Arthrobacter sp. Infecting Bacteriophage with Completely Sequenced Genome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111230

    Restriction digestion patterns of phage ArV2 genomic DNA. Lanes: 1 – ArV2 gDNA (nonrestricted); 2 – EcoRII; 3 – NotI; 4 – MboI; 5 – BamHI; 6 – Marker GeneRulerTM DNA Ladder Mix (Thermo Fisher Scientific).
    Figure Legend Snippet: Restriction digestion patterns of phage ArV2 genomic DNA. Lanes: 1 – ArV2 gDNA (nonrestricted); 2 – EcoRII; 3 – NotI; 4 – MboI; 5 – BamHI; 6 – Marker GeneRulerTM DNA Ladder Mix (Thermo Fisher Scientific).

    Techniques Used: Marker

    14) Product Images from "Cloning, expression and purification of the factor H binding protein and its interaction with factor H"

    Article Title: Cloning, expression and purification of the factor H binding protein and its interaction with factor H

    Journal: Iranian Journal of Microbiology

    doi:

    Confirmation of gene cloning: Lane 1, specific PCR product of fhbp gene; Lane 2, double digestion of recombinant plasmid by BamHI and SalI restriction enzymes ; Lane M, 100bp DNA ladder; lane 3, fhbp gene PCR product by plasmid universal primers; Lane 4, universal PCR product of pET 28a (+) as negative control.
    Figure Legend Snippet: Confirmation of gene cloning: Lane 1, specific PCR product of fhbp gene; Lane 2, double digestion of recombinant plasmid by BamHI and SalI restriction enzymes ; Lane M, 100bp DNA ladder; lane 3, fhbp gene PCR product by plasmid universal primers; Lane 4, universal PCR product of pET 28a (+) as negative control.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Recombinant, Plasmid Preparation, Positron Emission Tomography, Negative Control

    15) Product Images from "Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum"

    Article Title: Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni037

    Example of a single BIBAC fingerprint generated with five restriction enzymes (the HaeIII fragment ends were not labeled) and four fluorescent-ddNTPs of the SNaPshot Multiplex Ready Reaction Mix on the ABI 3100 DNA analyzer. The blue peaks show the BamHI fragments labeled with ddGTP-dR110; the green peaks show the HindIII fragments labeled with ddATP-dR6G; the black peaks show the XbaI fragments labeled with ddCTP–dTAMRA; the red peaks show the XhoI fragments labeled with ddTTP–dROX; and the orange peaks show the internal size standard LIZ-500.
    Figure Legend Snippet: Example of a single BIBAC fingerprint generated with five restriction enzymes (the HaeIII fragment ends were not labeled) and four fluorescent-ddNTPs of the SNaPshot Multiplex Ready Reaction Mix on the ABI 3100 DNA analyzer. The blue peaks show the BamHI fragments labeled with ddGTP-dR110; the green peaks show the HindIII fragments labeled with ddATP-dR6G; the black peaks show the XbaI fragments labeled with ddCTP–dTAMRA; the red peaks show the XhoI fragments labeled with ddTTP–dROX; and the orange peaks show the internal size standard LIZ-500.

    Techniques Used: Generated, Labeling, Multiplex Assay

    16) Product Images from "Borrelia burgdorferi Requires Glycerol for Maximum Fitness During The Tick Phase of the Enzootic Cycle"

    Article Title: Borrelia burgdorferi Requires Glycerol for Maximum Fitness During The Tick Phase of the Enzootic Cycle

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002102

    Confirmation of glpD disruption. A ) Southern blot analysis confirms the disruption of glpD . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI and blotted on a nylon membrane. Digoxygenein-11-dUTP labeled bb0243 probe was utilized to visualize glpD . Migration positions of DNA molecular size markers are indicated on the left. B ) glpD disruption occurred via by a double crossover insertion of flgB - aadA . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI or EcoRI, which would be specific for the proximal or distal bb0243 flanking chromosomal regions, respectively, and blotted to a nylon membrane. Blots were developed with a digoxygenein-11-dUTP-labeled aadA probe. A schematic diagram indicates the sizes of the fragments expected to contain flgB - aadA . Migration positions of DNA molecular size markers are indicated on the left of each panel.
    Figure Legend Snippet: Confirmation of glpD disruption. A ) Southern blot analysis confirms the disruption of glpD . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI and blotted on a nylon membrane. Digoxygenein-11-dUTP labeled bb0243 probe was utilized to visualize glpD . Migration positions of DNA molecular size markers are indicated on the left. B ) glpD disruption occurred via by a double crossover insertion of flgB - aadA . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI or EcoRI, which would be specific for the proximal or distal bb0243 flanking chromosomal regions, respectively, and blotted to a nylon membrane. Blots were developed with a digoxygenein-11-dUTP-labeled aadA probe. A schematic diagram indicates the sizes of the fragments expected to contain flgB - aadA . Migration positions of DNA molecular size markers are indicated on the left of each panel.

    Techniques Used: Southern Blot, Labeling, Migration

    17) Product Images from "Construction of a Novel DNA Vaccine Candidate Encoding an HspX-PPE44-EsxV Fusion Antigen of Mycobacterium tuberculosis"

    Article Title: Construction of a Novel DNA Vaccine Candidate Encoding an HspX-PPE44-EsxV Fusion Antigen of Mycobacterium tuberculosis

    Journal: Reports of Biochemistry & Molecular Biology

    doi:

    Colony-PCR and digestion products visualized on 1.2% agarose gels. A: Agarose gel electrophoresis of colony-PCR product. Lane 1: 1968 bp PCR product of fusion fragments, M: 100 bp-plus DNA size marker; B: BamHI and Xba XbaI digestion products of recombinant
    Figure Legend Snippet: Colony-PCR and digestion products visualized on 1.2% agarose gels. A: Agarose gel electrophoresis of colony-PCR product. Lane 1: 1968 bp PCR product of fusion fragments, M: 100 bp-plus DNA size marker; B: BamHI and Xba XbaI digestion products of recombinant

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Recombinant

    Agarose gels of recombinant vector. a: digestion of pGH/HspX-PPE44-EsxV by BamHI and XbaI . lane 1: pGH (2940 bp) and HspX-PPE4-EsxV (1968 bp); lane; M: 1kb DNA size marker. B: Ligated pcDNA3.1 (+)/hspX-ppe44-esxV. The band is Recombinant pcDNA3.1 (+)
    Figure Legend Snippet: Agarose gels of recombinant vector. a: digestion of pGH/HspX-PPE44-EsxV by BamHI and XbaI . lane 1: pGH (2940 bp) and HspX-PPE4-EsxV (1968 bp); lane; M: 1kb DNA size marker. B: Ligated pcDNA3.1 (+)/hspX-ppe44-esxV. The band is Recombinant pcDNA3.1 (+)

    Techniques Used: Recombinant, Plasmid Preparation, Marker

    Schematic map of the pcDNA3.1 (+)/HspX-PPE44-EsxV-His plasmid. The fusion segment consisting of hspX, linker, ppe44, linker, esxV sequences, and a 6-polyhistidine-tag was designed between the BamHI and XbaI restriction sites of pcDNA3.1 (+) downstream
    Figure Legend Snippet: Schematic map of the pcDNA3.1 (+)/HspX-PPE44-EsxV-His plasmid. The fusion segment consisting of hspX, linker, ppe44, linker, esxV sequences, and a 6-polyhistidine-tag was designed between the BamHI and XbaI restriction sites of pcDNA3.1 (+) downstream

    Techniques Used: Plasmid Preparation

    18) Product Images from "A Virus-Like Particle Vaccine Elicits Broad Neutralizing Antibody Responses in Humans to All Chikungunya Virus Genotypes"

    Article Title: A Virus-Like Particle Vaccine Elicits Broad Neutralizing Antibody Responses in Humans to All Chikungunya Virus Genotypes

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiw431

    Production of infectious Semliki Forest virus (SFV)–green fluorescent protein (GFP)–chikungunya virus (CHIKV) chimeric viruses. A , Genomic organization of alphaviruses (top). A modified SFV replicon (pSFV-GFP-BB) consisting of a truncated SFV genome under the transcriptional control of the cytomegalovirus promoter (bottom). The hepatitis delta virus ribozyme and polyadenylation sequence of SV40 were introduced to create the 3′ terminus of the genome. BamHI and AscI endonuclease recognition sites were introduced downstream of the 26S subgenomic promoter to allow introduction of structural gene fragments from multiple CHIKV strains. A GFP reporter gene was introduced under a second 26S subgenomic promoter to allow infection to be scored by GFP expression. B , Crystal structure of the CHIKV heterotrimer of E3 (gray ribbon), E2 (cyan ribbon), and E1 (green ribbon) proteins (PDB 3N44). Blue and black spheres highlight variation at amino acid residues found in 1 or > 1 of the 9 CHIKV strains tested, respectively. C , SFV-GFP-CHIKV viruses collected 72 hours after transfection of HEK-293T cells with pSFV-GFP-BB only or with pSFV-GFP-BB containing CHIKV structural genes from 1 of 9 different strains were serially diluted and used to infect Vero cells to determine infectivity, as measured by the percentage of GFP-positive cells. Error bars indicate the range of infectivity from duplicate wells. Data are representative of 5 independent experiments performed with independent virus preparations. D , Growth kinetics of SFV-GFP-CHIKV viruses. Vero cells were infected with SFV-GFP-CHIKV at a multiplicity of infection of 0.05. Virus-containing supernatant collected at the indicated times was serially diluted and used to infect Vero cells to determine virus titer. Shown are average titers, reported as infectious units/milliliter, obtained from 2 independent experiments performed in duplicate with independent virus stocks. Errors bars represent the range of infection. Abbreviation: UTR, untranslated region.
    Figure Legend Snippet: Production of infectious Semliki Forest virus (SFV)–green fluorescent protein (GFP)–chikungunya virus (CHIKV) chimeric viruses. A , Genomic organization of alphaviruses (top). A modified SFV replicon (pSFV-GFP-BB) consisting of a truncated SFV genome under the transcriptional control of the cytomegalovirus promoter (bottom). The hepatitis delta virus ribozyme and polyadenylation sequence of SV40 were introduced to create the 3′ terminus of the genome. BamHI and AscI endonuclease recognition sites were introduced downstream of the 26S subgenomic promoter to allow introduction of structural gene fragments from multiple CHIKV strains. A GFP reporter gene was introduced under a second 26S subgenomic promoter to allow infection to be scored by GFP expression. B , Crystal structure of the CHIKV heterotrimer of E3 (gray ribbon), E2 (cyan ribbon), and E1 (green ribbon) proteins (PDB 3N44). Blue and black spheres highlight variation at amino acid residues found in 1 or > 1 of the 9 CHIKV strains tested, respectively. C , SFV-GFP-CHIKV viruses collected 72 hours after transfection of HEK-293T cells with pSFV-GFP-BB only or with pSFV-GFP-BB containing CHIKV structural genes from 1 of 9 different strains were serially diluted and used to infect Vero cells to determine infectivity, as measured by the percentage of GFP-positive cells. Error bars indicate the range of infectivity from duplicate wells. Data are representative of 5 independent experiments performed with independent virus preparations. D , Growth kinetics of SFV-GFP-CHIKV viruses. Vero cells were infected with SFV-GFP-CHIKV at a multiplicity of infection of 0.05. Virus-containing supernatant collected at the indicated times was serially diluted and used to infect Vero cells to determine virus titer. Shown are average titers, reported as infectious units/milliliter, obtained from 2 independent experiments performed in duplicate with independent virus stocks. Errors bars represent the range of infection. Abbreviation: UTR, untranslated region.

    Techniques Used: Modification, Sequencing, Infection, Expressing, Transfection

    19) Product Images from "Francisella tularensis Molecular Typing Using Differential Insertion Sequence Amplification ▿"

    Article Title: Francisella tularensis Molecular Typing Using Differential Insertion Sequence Amplification ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00033-11

    Dendrogram of PFGE patterns obtained with BamHI-digested F. tularensis subsp. tularensis subtype A.I (A) as well as F. tularensis subsp. tularensis subtype A.II and F. tularensis subsp. holarctica type B (B). A representative BamHI-digested F. tularensis
    Figure Legend Snippet: Dendrogram of PFGE patterns obtained with BamHI-digested F. tularensis subsp. tularensis subtype A.I (A) as well as F. tularensis subsp. tularensis subtype A.II and F. tularensis subsp. holarctica type B (B). A representative BamHI-digested F. tularensis

    Techniques Used:

    20) Product Images from "An Improved Whole-Blood Gamma Interferon Assay Based on the CFP21-MPT64 Fusion Protein ▿"

    Article Title: An Improved Whole-Blood Gamma Interferon Assay Based on the CFP21-MPT64 Fusion Protein ▿

    Journal:

    doi: 10.1128/CVI.00486-08

    Expression and purification of rCM fusion protein. (A) Map of pPro2164 and the fusion gene encoding the predicted mature forms of CFP21 and MPT64, with a 45-bp linker, which was cloned into the BamHI and HindIII sites of pProExHTb, resulting in the recombinant
    Figure Legend Snippet: Expression and purification of rCM fusion protein. (A) Map of pPro2164 and the fusion gene encoding the predicted mature forms of CFP21 and MPT64, with a 45-bp linker, which was cloned into the BamHI and HindIII sites of pProExHTb, resulting in the recombinant

    Techniques Used: Expressing, Purification, Clone Assay, Recombinant

    21) Product Images from "Cre Recombination Mediated Cassette Exchange For Building Versatile Transgenic Human ESC lines"

    Article Title: Cre Recombination Mediated Cassette Exchange For Building Versatile Transgenic Human ESC lines

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1002/stem.38

    Replacement of the master GFP cassette with the targeting RFP cassette through RMCE. (A) Scheme of cassette exchange before and after RMCE. The sizes of DNA fragments from PCR and Southern blotting analysis were indicated. B, BamHI site. The correctly
    Figure Legend Snippet: Replacement of the master GFP cassette with the targeting RFP cassette through RMCE. (A) Scheme of cassette exchange before and after RMCE. The sizes of DNA fragments from PCR and Southern blotting analysis were indicated. B, BamHI site. The correctly

    Techniques Used: Polymerase Chain Reaction, Southern Blot

    22) Product Images from "The DNA Methylome of the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius"

    Article Title: The DNA Methylome of the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00137

    Identification of methylated recognition sequences in S. acidocaldarius genomic DNA by digestion assays. Restriction enzymes BsuRI (A) and BamHI, DpnI, and MboI (B) were used to highlight the presence/absence of methylated 5′-GGCC-3′ and 5′-GATC-3′ palindromes, respectively, in genomic DNA of S. acidocaldarius . Two types of substrates were digested: genomic DNA (gDNA) and a whole genome amplification of the genomic DNA (WGA). WGA is identical to gDNA in terms of nucleic sequence but it does not contain epigenetic marks (see material and methods). This negative control is only used in (A) . The addition of different restriction enzymes is symbolized by a positive sign “+” while reaction mix without restriction enzyme is represented by a negative sign “–”. Molecular size markers (GeneRuler DNA Ladder, Thermo Scientific) were loaded in both gels (M). Digestion patterns were obtained in 0.8% agarose gel stained with ethidium bromide and visualized under UV light.
    Figure Legend Snippet: Identification of methylated recognition sequences in S. acidocaldarius genomic DNA by digestion assays. Restriction enzymes BsuRI (A) and BamHI, DpnI, and MboI (B) were used to highlight the presence/absence of methylated 5′-GGCC-3′ and 5′-GATC-3′ palindromes, respectively, in genomic DNA of S. acidocaldarius . Two types of substrates were digested: genomic DNA (gDNA) and a whole genome amplification of the genomic DNA (WGA). WGA is identical to gDNA in terms of nucleic sequence but it does not contain epigenetic marks (see material and methods). This negative control is only used in (A) . The addition of different restriction enzymes is symbolized by a positive sign “+” while reaction mix without restriction enzyme is represented by a negative sign “–”. Molecular size markers (GeneRuler DNA Ladder, Thermo Scientific) were loaded in both gels (M). Digestion patterns were obtained in 0.8% agarose gel stained with ethidium bromide and visualized under UV light.

    Techniques Used: Methylation, Whole Genome Amplification, Sequencing, Negative Control, Agarose Gel Electrophoresis, Staining

    23) Product Images from "A Type II Protein Secretory Pathway Required for Levansucrase Secretion by Gluconacetobacter diazotrophicus"

    Article Title: A Type II Protein Secretory Pathway Required for Levansucrase Secretion by Gluconacetobacter diazotrophicus

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.186.15.5031-5039.2004

    Localization of the region of the library cosmid p21R1 complementing LsdA secretion in mutant M31. lsd genes are represented by arrows. lsdA and lsdB encode levansucrase and levanase, respectively. lsdX , -G , and - O are the first genes of the type II secretion operon. The ability of each plasmid to restore LsdA secretion is indicated in parentheses. Plasmid pALS118 encodes a mutated LsdG (C126G). Restriction sites: E, EcoRI; B, BamHI; Bg, BglII.
    Figure Legend Snippet: Localization of the region of the library cosmid p21R1 complementing LsdA secretion in mutant M31. lsd genes are represented by arrows. lsdA and lsdB encode levansucrase and levanase, respectively. lsdX , -G , and - O are the first genes of the type II secretion operon. The ability of each plasmid to restore LsdA secretion is indicated in parentheses. Plasmid pALS118 encodes a mutated LsdG (C126G). Restriction sites: E, EcoRI; B, BamHI; Bg, BglII.

    Techniques Used: Mutagenesis, Plasmid Preparation

    24) Product Images from "Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b"

    Article Title: Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b

    Journal: SpringerPlus

    doi: 10.1186/2193-1801-2-264

    Restriction analysis of expression vectors containing human interferon alpha 2b gene. Lane 1–2 pET28a-IAS digested with NdeI BamHI . Two bands corresponding to 5.369 kb pET28a(+) expression vector and 567 bp insert are indicated by arrows, Lane 3 DNA size marker.
    Figure Legend Snippet: Restriction analysis of expression vectors containing human interferon alpha 2b gene. Lane 1–2 pET28a-IAS digested with NdeI BamHI . Two bands corresponding to 5.369 kb pET28a(+) expression vector and 567 bp insert are indicated by arrows, Lane 3 DNA size marker.

    Techniques Used: Expressing, Plasmid Preparation, Marker

    Agarose gel (1%) electrophoretic analysis of pTA-IFN vector restriction digestion. Lane 1 DNA size marker, Lane 2–4 Restriction digestion of pTA-IFN vector from three different clones with NdeI and BamHI restriction enzymes. Two bands corresponding to 2.886 kb pTZ57R/T vector and 567 bp insert are indicated by arrows. Lane 5, hIFNα-2b gene, Lane 6 Undigested pTA-IFN vector.
    Figure Legend Snippet: Agarose gel (1%) electrophoretic analysis of pTA-IFN vector restriction digestion. Lane 1 DNA size marker, Lane 2–4 Restriction digestion of pTA-IFN vector from three different clones with NdeI and BamHI restriction enzymes. Two bands corresponding to 2.886 kb pTZ57R/T vector and 567 bp insert are indicated by arrows. Lane 5, hIFNα-2b gene, Lane 6 Undigested pTA-IFN vector.

    Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Marker, Clone Assay

    25) Product Images from "Identification of a Naegleria fowleri Membrane Protein Reactive with Anti-Human CD59 Antibody"

    Article Title: Identification of a Naegleria fowleri Membrane Protein Reactive with Anti-Human CD59 Antibody

    Journal:

    doi: 10.1128/IAI.74.2.1189-1195.2006

    Southern blot analysis of pathogenic and nonpathogenic Naegleria genomic DNAs (10 μg) hybridized with human CD59 cDNA. Genomic DNA was digested overnight with restriction enzyme EcoRI (A), BamHI (B), or HindIII (C); separated by gel electrophoresis;
    Figure Legend Snippet: Southern blot analysis of pathogenic and nonpathogenic Naegleria genomic DNAs (10 μg) hybridized with human CD59 cDNA. Genomic DNA was digested overnight with restriction enzyme EcoRI (A), BamHI (B), or HindIII (C); separated by gel electrophoresis;

    Techniques Used: Southern Blot, Nucleic Acid Electrophoresis

    26) Product Images from "Cutaneous human papillomavirus 88: Remarkable differences in viral load"

    Article Title: Cutaneous human papillomavirus 88: Remarkable differences in viral load

    Journal: International journal of cancer. Journal international du cancer

    doi: 10.1002/ijc.23115

    Direct visualization of HPV88 on an EtBr-stained gel, after digestion with BamHI, lane 1. Lane M, Gene Ruler 1 kb DNA ladder (Fermentas).
    Figure Legend Snippet: Direct visualization of HPV88 on an EtBr-stained gel, after digestion with BamHI, lane 1. Lane M, Gene Ruler 1 kb DNA ladder (Fermentas).

    Techniques Used: Staining

    27) Product Images from "qDSB-Seq is a general method for genome-wide quantification of DNA double-strand breaks using sequencing"

    Article Title: qDSB-Seq is a general method for genome-wide quantification of DNA double-strand breaks using sequencing

    Journal: Nature Communications

    doi: 10.1038/s41467-019-10332-8

    Quantitative DSB sequencing (qDSB-Seq) method. a A comparison of current DNA double-strand break (DSB) counting (e.g., immunofluorescence microscopy, quantitative PCR (qPCR)) and DSB sequencing strategies (e.g., BLESS 3 , i-BLESS 15 , END-Seq 6 , Break-Seq 4 , DSBCapture 5 ) with our qDSB-Seq method. b In qDSB-Seq protocol after DSB induction cells are treated with a restriction enzyme to introduce site-specific, infrequent DSBs (spike-ins). Next, DSBs are labeled (here using i-BLESS 15 or BLESS 3 ) and sequenced. Simultaneously, genomic DNA (gDNA) sequencing (or qPCR) is performed and used to estimate the cutting efficiency of the enzyme, and thus frequency of induced spike-in DSBs, which is then used to quantify the absolute DSB frequencies (per cell) of studied DSBs in the sample (Methods). c qDSB-Seq can be combined with any sequencing-based DSB labeling method. d , e Spike-in DSBs were induced in two different ways: d the studied cells were digested using the NotI, SrfI, AsiSI, or BamHI restriction enzyme in vitro; e cells expressing a restriction enzyme in vivo were mixed with the studied cells (I-SceI digestion) or alternatively a restriction enzyme was expressed in vivo in all studied cells (DIvA cells discussed below)
    Figure Legend Snippet: Quantitative DSB sequencing (qDSB-Seq) method. a A comparison of current DNA double-strand break (DSB) counting (e.g., immunofluorescence microscopy, quantitative PCR (qPCR)) and DSB sequencing strategies (e.g., BLESS 3 , i-BLESS 15 , END-Seq 6 , Break-Seq 4 , DSBCapture 5 ) with our qDSB-Seq method. b In qDSB-Seq protocol after DSB induction cells are treated with a restriction enzyme to introduce site-specific, infrequent DSBs (spike-ins). Next, DSBs are labeled (here using i-BLESS 15 or BLESS 3 ) and sequenced. Simultaneously, genomic DNA (gDNA) sequencing (or qPCR) is performed and used to estimate the cutting efficiency of the enzyme, and thus frequency of induced spike-in DSBs, which is then used to quantify the absolute DSB frequencies (per cell) of studied DSBs in the sample (Methods). c qDSB-Seq can be combined with any sequencing-based DSB labeling method. d , e Spike-in DSBs were induced in two different ways: d the studied cells were digested using the NotI, SrfI, AsiSI, or BamHI restriction enzyme in vitro; e cells expressing a restriction enzyme in vivo were mixed with the studied cells (I-SceI digestion) or alternatively a restriction enzyme was expressed in vivo in all studied cells (DIvA cells discussed below)

    Techniques Used: Sequencing, Immunofluorescence, Microscopy, Real-time Polymerase Chain Reaction, Introduce, Labeling, In Vitro, Expressing, In Vivo

    Related Articles

    Ligation:

    Article Title: Immobilized Cobalt Affinity Chromatography Provides a Novel, Efficient Method for Herpes Simplex Virus Type 1 Gene Vector Purification
    Article Snippet: .. The oligonucleotides (10 pM) were mixed in phosphate-buffered saline (PBS), denatured at 100°C for 10 min, and then annealed for 1 h. BamHI-digested plasmid pTZ18UgBpK− DNA was dephosphorylated, gel purified, and then ligated with the annealed HAT-encoding oligonucleotide mixture, and then the ligation mixture was transformed into Escherichia coli DH5α competent cells (Invitrogen). .. Single colonies from the transformation were cultured, and plasmid DNAs were extracted by use of a Quick plasmid kit (Qiagen, Valencia, Calif.).

    Cell Culture:

    Article Title: Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System
    Article Snippet: .. The pBlue Script II containing fusion truncated ORF2-NSP4 was transformed in E.coli DH5α, cultured in LB agar containing ampicillin (50 mg/L), then plasmid was extracted and double digested by BamHI and SalI restriction enzymes (Thermo Scientific, USA). .. Simultaneously, PfastBac1 plasmid was also double digested by BamHI and Sal1.

    Purification:

    Article Title: Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance
    Article Snippet: .. The purified plasmid was confirmed by single and double-digestion reactions by BamHI and XbaI restriction enzymes (Thermo Scientific, Waltham, MA, USA) and after confirmation was used to clone and express the left and right ZFN arrays. .. ZFN Cloning in pP15A, kanaR The nucleotide sequences coding for the left and right ZFP arrays were downloaded from http://zifit.partners.org/ZiFiT.

    Article Title: Immobilized Cobalt Affinity Chromatography Provides a Novel, Efficient Method for Herpes Simplex Virus Type 1 Gene Vector Purification
    Article Snippet: .. The oligonucleotides (10 pM) were mixed in phosphate-buffered saline (PBS), denatured at 100°C for 10 min, and then annealed for 1 h. BamHI-digested plasmid pTZ18UgBpK− DNA was dephosphorylated, gel purified, and then ligated with the annealed HAT-encoding oligonucleotide mixture, and then the ligation mixture was transformed into Escherichia coli DH5α competent cells (Invitrogen). .. Single colonies from the transformation were cultured, and plasmid DNAs were extracted by use of a Quick plasmid kit (Qiagen, Valencia, Calif.).

    Real-time Polymerase Chain Reaction:

    Article Title: Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice
    Article Snippet: .. Quantitative analysis of EBV BamHI W fragment DNA was performed by TaqMan (Applied Biosystems) quantitative PCR (qPCR) as described previously [ , ]. .. Reactions were run in duplicate on an ABI Prism 7700 Sequence Detector (Applied Biosystems).

    Generated:

    Article Title: Human Mre11/Human Rad50/Nbs1 and DNA Ligase III?/XRCC1 Protein Complexes Act Together in an Alternative Nonhomologous End Joining Pathway *
    Article Snippet: .. Linear DNA molecules were generated by digestion of pGADT7 (Invitrogen) with BamHI and EcoRI for the four nucleotide 5′ overhangs and of pcDNA4HisMax C (Invitrogen) with KpnI and PstI for the four nucleotide 3′ overhangs. .. Intramolecular joining of the incompatible ends was carried out in 25 m m MOPS (pH 7.0), 60 m m KCl, 0.2% Tween 20, 2 m m DTT, 4 m m MgCl2 , 2 m m MnCl2 , 0.5 m m ATP, 0.8 pmol of plasmid DNA, 10% polyethylene glycol, 0.01 pmol of DNA ligase III/XRCC1 or DNA ligase IV/XRCC4, and 0.06 pmol of hMre11 or MRN as indicated, in a volume of 10 μl.

    HAT Assay:

    Article Title: Immobilized Cobalt Affinity Chromatography Provides a Novel, Efficient Method for Herpes Simplex Virus Type 1 Gene Vector Purification
    Article Snippet: .. The oligonucleotides (10 pM) were mixed in phosphate-buffered saline (PBS), denatured at 100°C for 10 min, and then annealed for 1 h. BamHI-digested plasmid pTZ18UgBpK− DNA was dephosphorylated, gel purified, and then ligated with the annealed HAT-encoding oligonucleotide mixture, and then the ligation mixture was transformed into Escherichia coli DH5α competent cells (Invitrogen). .. Single colonies from the transformation were cultured, and plasmid DNAs were extracted by use of a Quick plasmid kit (Qiagen, Valencia, Calif.).

    Transformation Assay:

    Article Title: Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System
    Article Snippet: .. The pBlue Script II containing fusion truncated ORF2-NSP4 was transformed in E.coli DH5α, cultured in LB agar containing ampicillin (50 mg/L), then plasmid was extracted and double digested by BamHI and SalI restriction enzymes (Thermo Scientific, USA). .. Simultaneously, PfastBac1 plasmid was also double digested by BamHI and Sal1.

    Article Title: Immobilized Cobalt Affinity Chromatography Provides a Novel, Efficient Method for Herpes Simplex Virus Type 1 Gene Vector Purification
    Article Snippet: .. The oligonucleotides (10 pM) were mixed in phosphate-buffered saline (PBS), denatured at 100°C for 10 min, and then annealed for 1 h. BamHI-digested plasmid pTZ18UgBpK− DNA was dephosphorylated, gel purified, and then ligated with the annealed HAT-encoding oligonucleotide mixture, and then the ligation mixture was transformed into Escherichia coli DH5α competent cells (Invitrogen). .. Single colonies from the transformation were cultured, and plasmid DNAs were extracted by use of a Quick plasmid kit (Qiagen, Valencia, Calif.).

    Plasmid Preparation:

    Article Title: Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System
    Article Snippet: .. The pBlue Script II containing fusion truncated ORF2-NSP4 was transformed in E.coli DH5α, cultured in LB agar containing ampicillin (50 mg/L), then plasmid was extracted and double digested by BamHI and SalI restriction enzymes (Thermo Scientific, USA). .. Simultaneously, PfastBac1 plasmid was also double digested by BamHI and Sal1.

    Article Title: Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance
    Article Snippet: .. The purified plasmid was confirmed by single and double-digestion reactions by BamHI and XbaI restriction enzymes (Thermo Scientific, Waltham, MA, USA) and after confirmation was used to clone and express the left and right ZFN arrays. .. ZFN Cloning in pP15A, kanaR The nucleotide sequences coding for the left and right ZFP arrays were downloaded from http://zifit.partners.org/ZiFiT.

    Article Title: Immobilized Cobalt Affinity Chromatography Provides a Novel, Efficient Method for Herpes Simplex Virus Type 1 Gene Vector Purification
    Article Snippet: .. The oligonucleotides (10 pM) were mixed in phosphate-buffered saline (PBS), denatured at 100°C for 10 min, and then annealed for 1 h. BamHI-digested plasmid pTZ18UgBpK− DNA was dephosphorylated, gel purified, and then ligated with the annealed HAT-encoding oligonucleotide mixture, and then the ligation mixture was transformed into Escherichia coli DH5α competent cells (Invitrogen). .. Single colonies from the transformation were cultured, and plasmid DNAs were extracted by use of a Quick plasmid kit (Qiagen, Valencia, Calif.).

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    Thermo Fisher bamhi
    A: pFastBac1 and pBlueScript-ORF2-NSP4 Genes is Double Digested With <t>BamHI</t> and <t>SalI</t> RESTRICTION ENZYMES A: The linearized pFastBac1 and ORF2-NSP4 respectively appeared at 4775 and 2000 bp. Lane 1: pFastBac1 is double digested with BamHI/SalI ; Lane 2: pBlueScript-ORF2-NSP4 is double digested with BamHI/SalI.B : ORF2-NSP4 gene was cloned in pFastBac1. Lane 1 - 2: pFastBac1-ORF2-NSP4; and Lane3 - 4: pFastBac1. C: the position site of ORF2-NSP4 was confirmed by single and double digestion and the length of subcloned genes after single digestion was 6775 bp while after double digestion was 4775 and 2000 bp. Lane 1 - 2: pFastBac1-ORF2-NSP4 single digestion BamHI ; Lane 3 - 4: pFastBac1-ORF2-NSP4 single digestion SalI; and Lane 5-6: pFastBac1-ORF2-NSP4 double digestion BamHI/SalI .
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    A: pFastBac1 and pBlueScript-ORF2-NSP4 Genes is Double Digested With BamHI and SalI RESTRICTION ENZYMES A: The linearized pFastBac1 and ORF2-NSP4 respectively appeared at 4775 and 2000 bp. Lane 1: pFastBac1 is double digested with BamHI/SalI ; Lane 2: pBlueScript-ORF2-NSP4 is double digested with BamHI/SalI.B : ORF2-NSP4 gene was cloned in pFastBac1. Lane 1 - 2: pFastBac1-ORF2-NSP4; and Lane3 - 4: pFastBac1. C: the position site of ORF2-NSP4 was confirmed by single and double digestion and the length of subcloned genes after single digestion was 6775 bp while after double digestion was 4775 and 2000 bp. Lane 1 - 2: pFastBac1-ORF2-NSP4 single digestion BamHI ; Lane 3 - 4: pFastBac1-ORF2-NSP4 single digestion SalI; and Lane 5-6: pFastBac1-ORF2-NSP4 double digestion BamHI/SalI .

    Journal: Jundishapur Journal of Microbiology

    Article Title: Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System

    doi: 10.5812/jjm.40303

    Figure Lengend Snippet: A: pFastBac1 and pBlueScript-ORF2-NSP4 Genes is Double Digested With BamHI and SalI RESTRICTION ENZYMES A: The linearized pFastBac1 and ORF2-NSP4 respectively appeared at 4775 and 2000 bp. Lane 1: pFastBac1 is double digested with BamHI/SalI ; Lane 2: pBlueScript-ORF2-NSP4 is double digested with BamHI/SalI.B : ORF2-NSP4 gene was cloned in pFastBac1. Lane 1 - 2: pFastBac1-ORF2-NSP4; and Lane3 - 4: pFastBac1. C: the position site of ORF2-NSP4 was confirmed by single and double digestion and the length of subcloned genes after single digestion was 6775 bp while after double digestion was 4775 and 2000 bp. Lane 1 - 2: pFastBac1-ORF2-NSP4 single digestion BamHI ; Lane 3 - 4: pFastBac1-ORF2-NSP4 single digestion SalI; and Lane 5-6: pFastBac1-ORF2-NSP4 double digestion BamHI/SalI .

    Article Snippet: The pBlue Script II containing fusion truncated ORF2-NSP4 was transformed in E.coli DH5α, cultured in LB agar containing ampicillin (50 mg/L), then plasmid was extracted and double digested by BamHI and SalI restriction enzymes (Thermo Scientific, USA).

    Techniques: Clone Assay

    REA of BoHV-1 isolates and reference strain by BamHI (A), HindIII (B), and EcoRI (C). Lane (1):DNA ladder (100 bp plus and 1kbp plus, ThermoScientific), Lane (2): KY748023 (KYS-73675-Milk), Lane (3): KY748022 (TGM-IZ41-Milk), Lane (4): KY748020 (ANK-SKR-Lng), Lane (5): KY748021 (Halk-THYM-Thm), Lane (6): Cooper strain. REA pattern differences indicated by *; KY748022 (Lane3) and KY748021 (Lane 5) were BoHV-1.2a while the other viruses in the test were BoHV-1.1 strains

    Journal: Tropical Animal Health and Production

    Article Title: Molecular and antigenic characterization of bovine herpesvirus type 1 (BoHV-1) strains from cattle with diverse clinical cases in Turkey

    doi: 10.1007/s11250-019-02042-6

    Figure Lengend Snippet: REA of BoHV-1 isolates and reference strain by BamHI (A), HindIII (B), and EcoRI (C). Lane (1):DNA ladder (100 bp plus and 1kbp plus, ThermoScientific), Lane (2): KY748023 (KYS-73675-Milk), Lane (3): KY748022 (TGM-IZ41-Milk), Lane (4): KY748020 (ANK-SKR-Lng), Lane (5): KY748021 (Halk-THYM-Thm), Lane (6): Cooper strain. REA pattern differences indicated by *; KY748022 (Lane3) and KY748021 (Lane 5) were BoHV-1.2a while the other viruses in the test were BoHV-1.1 strains

    Article Snippet: For REA, fastdigest Hind III, BamHI, and EcoRI (ThermoScientific, USA) restriction enzymes were used according to the manufacturer’s instructions.

    Techniques:

    Confirmation of Self-Ligated pP15A, kana R by Digestion Lane 2: digestion of pP15A, kana R vector with BamHI ; lane 3, xbaI; Lane 4, double digestion with BamHI-XbaI; Lane 5, undigested pP15A, kana R vector; Lanes 1 and 6, GeneRuler DNA ladder mix (Thermo Scientific, Waltham, MA, USA).

    Journal: Jundishapur Journal of Microbiology

    Article Title: Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance

    doi: 10.5812/jjm.29384

    Figure Lengend Snippet: Confirmation of Self-Ligated pP15A, kana R by Digestion Lane 2: digestion of pP15A, kana R vector with BamHI ; lane 3, xbaI; Lane 4, double digestion with BamHI-XbaI; Lane 5, undigested pP15A, kana R vector; Lanes 1 and 6, GeneRuler DNA ladder mix (Thermo Scientific, Waltham, MA, USA).

    Article Snippet: The purified plasmid was confirmed by single and double-digestion reactions by BamHI and XbaI restriction enzymes (Thermo Scientific, Waltham, MA, USA) and after confirmation was used to clone and express the left and right ZFN arrays.

    Techniques: Plasmid Preparation

    Digital imprinting of dsRNA recognition and processing on a ds[RNA-DNA] matrix. Shown in (a) and (d) is the sequence of the 39-bp ds[RNA-DNA] (12-bp of dsRNA, 27-bp of dsDNA), containing an A. aeolicus RNase III processing site positioned in the middle of the dsRNA segment (|dsRNA 0 〉. The dsRNA sequence corresponds to the duplex stem in the A. aeolicus 23S rRNA precursor 38 . The red rectangles indicate sites of enzyme contact 38 , and the scissile phosphodiesters are indicated by the red arrows. (a) Input 1 (red): RNase III cleavage of the target site would create a surface-attached, 23-bp ds[RNA-DNA] with a 2-nt, 3′-overhang terminus (calculated length ~7.1 nm). Input 2 (magenta), uses the RNase III E110A mutant, which retains dsRNA-binding ability 31 39 . This input does not alter the height, and is essentially identical to the control experiment that omits RNase III (Input 3: green) (see also Table 1 ). (b) The H OUT /H IN value indicates that Output 1 corresponds to a ds[RNA-DNA] that is processed by RNase III (red dashed line), whereas Outputs 2 and 3 correspond to an unaltered dsDNA-dsRNA segment (black dashed line). (c) schematically depicts the output matrices [1–3], in which [1] consists of a monolayer of duplexes comprising the unaltered |dsDNA 0 〉 segment and a cleaved |dsRNA 0 〉 segment. The structures in (d) highlight the BamHI recognition sequence (enclosed in blue rectangles). BamHI action would create a surface-bound, 4-bp product possessing a 4-nt 3′-overhang (calculated length ~1.4 nm). In (e), three distinct ‘imprints’ are generated when BamHI is included (Inputs 1+–3+). For Input 1+, the ds[RNA-DNA] is processed at the BamHI site (blue dashed line), when RNase III also is present. For Input 3+, BamHI displays only limited catalytic action in the absence of RNase III, while for Input 2+, BamHI is inhibited in the presence of the E110A RNase III mutant (note error bars for Outputs 2+ and 3+). (f) schematically depicts the output matrices [1–3+], in which [1+] consists of a monolayer of duplexes comprising the cleaved |dsDNA 0 〉 segment, and [3+] consists of a mixed monolayer of cleaved |dsDNA 0 〉 segments and intact duplexes. Data are means, including the standard deviations.

    Journal: Scientific Reports

    Article Title: Digital Imprinting of RNA Recognition and Processing on a Self-Assembled Nucleic Acid Matrix

    doi: 10.1038/srep02550

    Figure Lengend Snippet: Digital imprinting of dsRNA recognition and processing on a ds[RNA-DNA] matrix. Shown in (a) and (d) is the sequence of the 39-bp ds[RNA-DNA] (12-bp of dsRNA, 27-bp of dsDNA), containing an A. aeolicus RNase III processing site positioned in the middle of the dsRNA segment (|dsRNA 0 〉. The dsRNA sequence corresponds to the duplex stem in the A. aeolicus 23S rRNA precursor 38 . The red rectangles indicate sites of enzyme contact 38 , and the scissile phosphodiesters are indicated by the red arrows. (a) Input 1 (red): RNase III cleavage of the target site would create a surface-attached, 23-bp ds[RNA-DNA] with a 2-nt, 3′-overhang terminus (calculated length ~7.1 nm). Input 2 (magenta), uses the RNase III E110A mutant, which retains dsRNA-binding ability 31 39 . This input does not alter the height, and is essentially identical to the control experiment that omits RNase III (Input 3: green) (see also Table 1 ). (b) The H OUT /H IN value indicates that Output 1 corresponds to a ds[RNA-DNA] that is processed by RNase III (red dashed line), whereas Outputs 2 and 3 correspond to an unaltered dsDNA-dsRNA segment (black dashed line). (c) schematically depicts the output matrices [1–3], in which [1] consists of a monolayer of duplexes comprising the unaltered |dsDNA 0 〉 segment and a cleaved |dsRNA 0 〉 segment. The structures in (d) highlight the BamHI recognition sequence (enclosed in blue rectangles). BamHI action would create a surface-bound, 4-bp product possessing a 4-nt 3′-overhang (calculated length ~1.4 nm). In (e), three distinct ‘imprints’ are generated when BamHI is included (Inputs 1+–3+). For Input 1+, the ds[RNA-DNA] is processed at the BamHI site (blue dashed line), when RNase III also is present. For Input 3+, BamHI displays only limited catalytic action in the absence of RNase III, while for Input 2+, BamHI is inhibited in the presence of the E110A RNase III mutant (note error bars for Outputs 2+ and 3+). (f) schematically depicts the output matrices [1–3+], in which [1+] consists of a monolayer of duplexes comprising the cleaved |dsDNA 0 〉 segment, and [3+] consists of a mixed monolayer of cleaved |dsDNA 0 〉 segments and intact duplexes. Data are means, including the standard deviations.

    Article Snippet: Preparation of RNA-DNA chimera sequences A thiolated, 39-nucleotide RNA-DNA chimera sequence containing an RNase III cleavage site within the 27-nt RNA segment and a BamHI restriction site within the 12-nt DNA segment (see Seq1 below), as well as the complementary, non-thiolated [RNA-DNA] sequence (see Seq2 below) were purchased from ThermoFisher Scientific in HPLC-purified form.

    Techniques: Sequencing, Mutagenesis, Binding Assay, Generated

    Density-dependent steric regulation of imprinting a ds[RNA-DNA] matrix. (a) The final heights (H OUT ) of six separate Inputs are dependent upon the initial height (H IN ) of the ds[RNA-DNA] matrix. Input 1 (with RNase III). Input 2 (with E110A). Input 3 (controls, either lacking RNase III or with RNase III without the catalytic cofactor, Mg 2+ ). Input 1+ (with RNase III and BamHI). Input 2+ (with E110A and BamHI). Input 3+ (with BamHI alone). All dashed lines in (a) relate the data points to a linear regression. The data for Output 1 show that RNase III can process the dsRNA segment regardless of ds[RNA-DNA] density, which is related to the initial height (see schematic representation on top). Outputs 2 and 3 are consistent with an unaltered ds[RNA-DNA] chimera (represented by the solid diagonal line: H OUT = H IN ). BamHI gains full access to its site in combination with RNase III (Output 1+) as H OUT 1+ ≪ H OUT 1 , while it is essentially completely blocked in combination with the E110A mutant (Output 2+) as H OUT 2+ ~ H IN . BamHI restriction enzyme efficiency acting alone (Input 3+) must be lower than that of RNase III alone (Input 1), as the height of an matrix consisting of ds[RNA-DNA] molecules cleaved by BamHI would be lower than the height of an matrix cleaved by RNase III, and, in contrast, H OUT 3+ > H OUT 1 for relatively dense matrices (H IN > 10 nm). Data are means, and include standard deviations. (b) Schematic depiction of the effect of different inputs on a highly dense ds[RNA-DNA] matrix, including a steric hindrance-based model that shows how the ‘imprint’ (Output n+) is a step (i.e. digital) function of Input n+ (n = 1,3), as shown in (a).

    Journal: Scientific Reports

    Article Title: Digital Imprinting of RNA Recognition and Processing on a Self-Assembled Nucleic Acid Matrix

    doi: 10.1038/srep02550

    Figure Lengend Snippet: Density-dependent steric regulation of imprinting a ds[RNA-DNA] matrix. (a) The final heights (H OUT ) of six separate Inputs are dependent upon the initial height (H IN ) of the ds[RNA-DNA] matrix. Input 1 (with RNase III). Input 2 (with E110A). Input 3 (controls, either lacking RNase III or with RNase III without the catalytic cofactor, Mg 2+ ). Input 1+ (with RNase III and BamHI). Input 2+ (with E110A and BamHI). Input 3+ (with BamHI alone). All dashed lines in (a) relate the data points to a linear regression. The data for Output 1 show that RNase III can process the dsRNA segment regardless of ds[RNA-DNA] density, which is related to the initial height (see schematic representation on top). Outputs 2 and 3 are consistent with an unaltered ds[RNA-DNA] chimera (represented by the solid diagonal line: H OUT = H IN ). BamHI gains full access to its site in combination with RNase III (Output 1+) as H OUT 1+ ≪ H OUT 1 , while it is essentially completely blocked in combination with the E110A mutant (Output 2+) as H OUT 2+ ~ H IN . BamHI restriction enzyme efficiency acting alone (Input 3+) must be lower than that of RNase III alone (Input 1), as the height of an matrix consisting of ds[RNA-DNA] molecules cleaved by BamHI would be lower than the height of an matrix cleaved by RNase III, and, in contrast, H OUT 3+ > H OUT 1 for relatively dense matrices (H IN > 10 nm). Data are means, and include standard deviations. (b) Schematic depiction of the effect of different inputs on a highly dense ds[RNA-DNA] matrix, including a steric hindrance-based model that shows how the ‘imprint’ (Output n+) is a step (i.e. digital) function of Input n+ (n = 1,3), as shown in (a).

    Article Snippet: Preparation of RNA-DNA chimera sequences A thiolated, 39-nucleotide RNA-DNA chimera sequence containing an RNase III cleavage site within the 27-nt RNA segment and a BamHI restriction site within the 12-nt DNA segment (see Seq1 below), as well as the complementary, non-thiolated [RNA-DNA] sequence (see Seq2 below) were purchased from ThermoFisher Scientific in HPLC-purified form.

    Techniques: Mutagenesis