phusion high fidelity dna polymerase  (New England Biolabs)


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    Structured Review

    New England Biolabs phusion high fidelity dna polymerase
    Cloned pre-mir-122 stem-loop region sequences from HepG2 <t>DNA</t> show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with <t>Phusion</t> high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells"

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122471

    Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.
    Figure Legend Snippet: Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.

    Techniques Used: Clone Assay, Amplification, Sequencing, Polymerase Chain Reaction, Variant Assay

    2) Product Images from "Global analysis of putative phospholipases in the malaria parasite Plasmodium falciparum reveals critical factors for parasite proliferation"

    Article Title: Global analysis of putative phospholipases in the malaria parasite Plasmodium falciparum reveals critical factors for parasite proliferation

    Journal: bioRxiv

    doi: 10.1101/2021.06.28.450158

    PNPLA2-KO parasites have a defect in the mtETC. A-G) Drug susceptibility assays of WT and PNPLA2-KO parasites using proguanil (A), atovaquone (B), myxothiazol (C), antimycin A (D), DSM1 (E), dihydroartemisinin (DHA, F), primaquine (G). Parasite growth was assessed by measuring DNA content using SYBR gold when exposed to varying concentrations of drugs for 96 h. The growth of DMSO-treated control parasites was set to 100%. Shown are means +/- SD of 3 to 6 independent experiments performed in duplicate. Calculated IC 50 values with 95% confidence intervals are shown below each graph. H) The artificial electron acceptor decylubiquinone (DCUQ) does not rescue growth of PNLA2-KO parasites. WT and PNPLA2-KO parasites were grown in presence of various concentrations of DCUQ for two parasites cycles and parasitemia was evaluated using flow cytometry. As positive control, WT parasites were additionally treated with 1,15 nM atovaquone. Shown are means +/- SD of three independent experiments. I) PNPLA2-KO parasites have a defect in sustaining normal ΔΨm. C2-arrested WT and PNPLA2-KO schizonts that had been treated with DMSO (solvent control), 200 nM or 1 µM of proguanil were stained with the mitochondrial potentiometric dye rhodamine123 (Rho123, green) and parasites with a strong, weak or absent mitochondrial rhodamine123 signal were quantified by fluorescence microscopy. Shown are means +/- SD of four independent experiments, in which a total of 352 to 414 schizonts were analyzed per cell line and condition. For statistical evaluation a one-way ANOVA followed by a Holm-Sidak multiple comparison test was performed (*p
    Figure Legend Snippet: PNPLA2-KO parasites have a defect in the mtETC. A-G) Drug susceptibility assays of WT and PNPLA2-KO parasites using proguanil (A), atovaquone (B), myxothiazol (C), antimycin A (D), DSM1 (E), dihydroartemisinin (DHA, F), primaquine (G). Parasite growth was assessed by measuring DNA content using SYBR gold when exposed to varying concentrations of drugs for 96 h. The growth of DMSO-treated control parasites was set to 100%. Shown are means +/- SD of 3 to 6 independent experiments performed in duplicate. Calculated IC 50 values with 95% confidence intervals are shown below each graph. H) The artificial electron acceptor decylubiquinone (DCUQ) does not rescue growth of PNLA2-KO parasites. WT and PNPLA2-KO parasites were grown in presence of various concentrations of DCUQ for two parasites cycles and parasitemia was evaluated using flow cytometry. As positive control, WT parasites were additionally treated with 1,15 nM atovaquone. Shown are means +/- SD of three independent experiments. I) PNPLA2-KO parasites have a defect in sustaining normal ΔΨm. C2-arrested WT and PNPLA2-KO schizonts that had been treated with DMSO (solvent control), 200 nM or 1 µM of proguanil were stained with the mitochondrial potentiometric dye rhodamine123 (Rho123, green) and parasites with a strong, weak or absent mitochondrial rhodamine123 signal were quantified by fluorescence microscopy. Shown are means +/- SD of four independent experiments, in which a total of 352 to 414 schizonts were analyzed per cell line and condition. For statistical evaluation a one-way ANOVA followed by a Holm-Sidak multiple comparison test was performed (*p

    Techniques Used: Flow Cytometry, Positive Control, Staining, Fluorescence, Microscopy

    3) Product Images from "Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells"

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122471

    Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.
    Figure Legend Snippet: Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.

    Techniques Used: Clone Assay, Amplification, Sequencing, Polymerase Chain Reaction, Variant Assay

    4) Product Images from "Post-Zygotic and Inter-Individual Structural Genetic Variation in a Presumptive Enhancer Element of the Locus between the IL10Rβ and IFNAR1 Genes"

    Article Title: Post-Zygotic and Inter-Individual Structural Genetic Variation in a Presumptive Enhancer Element of the Locus between the IL10Rβ and IFNAR1 Genes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067752

    Variable length of alleles within hypervariable region showing post-zygotic variation. Panel A shows post-zygotic mosaicism in healthy and phenotypically concordant monozygotic twin pair 148341/148342, with five alleles observed in twin 148341, and three alleles present in co-twin 148342. Similarly, panel B displays post-zygotic variation in another monozygotic twin pair 004_01/004_02. In total 5 different alleles are shown on this gel and only one of them is overlapping between both twins. Panel C illustrates post-zygotic mosaicism in breast cancer patient SK58. There are three different alleles in DNA from morphologically normal breast tissue (UM), two alleles in blood cells (BL) and three alleles in primary tumor (PT). In panels A , B and C , Taq DNA polymerase was used for initial PCR amplification from genomic DNA, as indicated by suffix “T” in the ID of each plasmid clone. In panel D , Phusion DNA polymerase confirmed post-zygotic mosaicism in monozygotic twin pair 148341/148342, as indicated by suffix “Ph” in the ID of each plasmid clone. The length of inserts in all plasmid clones was estimated after EcoRI digestion releasing the insert, and using 1% agarose gel. BL, PT and UM indicate peripheral blood DNA, primary breast tumor and healthy morphologically normal breast tissue from a patient affected with breast cancer, respectively.
    Figure Legend Snippet: Variable length of alleles within hypervariable region showing post-zygotic variation. Panel A shows post-zygotic mosaicism in healthy and phenotypically concordant monozygotic twin pair 148341/148342, with five alleles observed in twin 148341, and three alleles present in co-twin 148342. Similarly, panel B displays post-zygotic variation in another monozygotic twin pair 004_01/004_02. In total 5 different alleles are shown on this gel and only one of them is overlapping between both twins. Panel C illustrates post-zygotic mosaicism in breast cancer patient SK58. There are three different alleles in DNA from morphologically normal breast tissue (UM), two alleles in blood cells (BL) and three alleles in primary tumor (PT). In panels A , B and C , Taq DNA polymerase was used for initial PCR amplification from genomic DNA, as indicated by suffix “T” in the ID of each plasmid clone. In panel D , Phusion DNA polymerase confirmed post-zygotic mosaicism in monozygotic twin pair 148341/148342, as indicated by suffix “Ph” in the ID of each plasmid clone. The length of inserts in all plasmid clones was estimated after EcoRI digestion releasing the insert, and using 1% agarose gel. BL, PT and UM indicate peripheral blood DNA, primary breast tumor and healthy morphologically normal breast tissue from a patient affected with breast cancer, respectively.

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

    5) Product Images from "DUX4 regulates oocyte to embryo transition in human"

    Article Title: DUX4 regulates oocyte to embryo transition in human

    Journal: bioRxiv

    doi: 10.1101/732289

    Interactions of DUX4. Microscale thermophoresis binding analysis of peptides to human KIX domain. (a) 9aaTAD peptide (C370-Q386) (b) KBM peptide (E414-E423) (c) KBM binding to KIX with saturating 9aaTAD. (d) Inter-HD interactions stabilizing DUX4 HD1 and HD2 in absence of bound DNA (e) Sequence comparison of HD1-HD2 interacting residues seen in human DUX4 with other primates and other human double HD transcription factors. (f) RMSF (Cα atoms) of X-ray structure of DUX4 with (red curve) and without (blue curve) bound DNAduring a 100 ns MD simulation. HD1 (blue), linker (magenta) and HD2 (gold). (g) RMSD (backbone atoms) with reference to starting conformation) of X-ray structure of DUX4 HD1-HD2 with and without bound DNA, and separately for HD1 and for HD2 with bound DNA, during 100 ns MD simulations. (h) Superposed conformations of DUX4 with (left) and without (right) DNA, sampled during 100 ns (top) and final 20 ns (bottom) of the simulation. Chain traces are colored based on the Cα-atom RMSD relative to the median structure at 50 ns or 90 ns. DNA-bound DUX4 shows higher stability than DNA-free DUX4; both exhibit larger fluctuations at the unconstrained N-termini and linker loops. A more stable conformation of DNA-free DUX4 exposing residues of the recognition helices was attained during the last 20 ns. (i) Final pose, DNA-free DUX4 (blue), after 100 ns simulation with HD1 superposed on HD1 of DNA-bound DUX4 X-ray structure (red and grey), revealing the degree of “opening” seen in the simulation; e.g. the Cα-atom of R146 of the third helix of HD2 differs in relative position by 38.6 Å.
    Figure Legend Snippet: Interactions of DUX4. Microscale thermophoresis binding analysis of peptides to human KIX domain. (a) 9aaTAD peptide (C370-Q386) (b) KBM peptide (E414-E423) (c) KBM binding to KIX with saturating 9aaTAD. (d) Inter-HD interactions stabilizing DUX4 HD1 and HD2 in absence of bound DNA (e) Sequence comparison of HD1-HD2 interacting residues seen in human DUX4 with other primates and other human double HD transcription factors. (f) RMSF (Cα atoms) of X-ray structure of DUX4 with (red curve) and without (blue curve) bound DNAduring a 100 ns MD simulation. HD1 (blue), linker (magenta) and HD2 (gold). (g) RMSD (backbone atoms) with reference to starting conformation) of X-ray structure of DUX4 HD1-HD2 with and without bound DNA, and separately for HD1 and for HD2 with bound DNA, during 100 ns MD simulations. (h) Superposed conformations of DUX4 with (left) and without (right) DNA, sampled during 100 ns (top) and final 20 ns (bottom) of the simulation. Chain traces are colored based on the Cα-atom RMSD relative to the median structure at 50 ns or 90 ns. DNA-bound DUX4 shows higher stability than DNA-free DUX4; both exhibit larger fluctuations at the unconstrained N-termini and linker loops. A more stable conformation of DNA-free DUX4 exposing residues of the recognition helices was attained during the last 20 ns. (i) Final pose, DNA-free DUX4 (blue), after 100 ns simulation with HD1 superposed on HD1 of DNA-bound DUX4 X-ray structure (red and grey), revealing the degree of “opening” seen in the simulation; e.g. the Cα-atom of R146 of the third helix of HD2 differs in relative position by 38.6 Å.

    Techniques Used: Microscale Thermophoresis, Binding Assay, Sequencing

    6) Product Images from "Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction"

    Article Title: Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction

    Journal: Current Protocols in Microbiology

    doi: 10.1002/cpmc.89

    PCR to test the efficiency of polymerases in amplifying PCR products from supernatants from different spore concentrations of the A. fumigatus wild‐type strain with primers ITS1/D2 (expected PCR band sizes is ∼1.2 kb). ( A ) Phusion High‐Fidelity DNA polymerase (New England Biolabs). ( B ) MyTaq RED Mix DNA polymerase (Bioline). P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).
    Figure Legend Snippet: PCR to test the efficiency of polymerases in amplifying PCR products from supernatants from different spore concentrations of the A. fumigatus wild‐type strain with primers ITS1/D2 (expected PCR band sizes is ∼1.2 kb). ( A ) Phusion High‐Fidelity DNA polymerase (New England Biolabs). ( B ) MyTaq RED Mix DNA polymerase (Bioline). P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).

    Techniques Used: Polymerase Chain Reaction, Amplification, Negative Control

    7) Product Images from "The Putative Endonuclease Activity of MutL Is Required for the Segmental Gene Conversion Events That Drive Antigenic Variation of the Lyme Disease Spirochete"

    Article Title: The Putative Endonuclease Activity of MutL Is Required for the Segmental Gene Conversion Events That Drive Antigenic Variation of the Lyme Disease Spirochete

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2022.888494

    Schematic of plasmid construction and introduction of point mutations into B. burgdorferi chromosomal mutL . (A) To generate the E. coli plasmids carrying point mutations in mutL we designed a two-insert strategy where two mutL double stranded fragments carrying complementary base pair changes (represented as an X) were ligated with each other and to pJET1.2 using the HiFi DNA Assembly mix from NEB. This assembly mix contains a 5′-3′ exonuclease that generates single stranded 3′ overhangs. These overhangs are able to anneal for a high-fidelity polymerase to extend the overhangs and fill in the gaps; finally, a DNA ligase seals the nicks. (B) The pJET plasmids containing the mutated versions of mutL were then digested with Xho I and Nco I, and (C) the mutated mutL inserts were then cloned into pOK12, (D) generating the plasmids pMC140, pMC141, pMC142, and pMC143 (see Supplementary Table 3 ). We then inserted the gentamicin resistance cassette with its own promoter (black arrow) downstream of mutL at the Nco I site. (E) Finally, we cloned the bb0212 gene downstream of the gent cassette to maintain the natural gene order in the B. burgdorferi chromosome. (F) Plasmids pMC148, pMC149, pMC150, and pMC151 were used to transform B. burgdorferi B31 wild-type clone 5A4 [20] and (G) GentR-KanS transformants were further analyzed for double recombination events.
    Figure Legend Snippet: Schematic of plasmid construction and introduction of point mutations into B. burgdorferi chromosomal mutL . (A) To generate the E. coli plasmids carrying point mutations in mutL we designed a two-insert strategy where two mutL double stranded fragments carrying complementary base pair changes (represented as an X) were ligated with each other and to pJET1.2 using the HiFi DNA Assembly mix from NEB. This assembly mix contains a 5′-3′ exonuclease that generates single stranded 3′ overhangs. These overhangs are able to anneal for a high-fidelity polymerase to extend the overhangs and fill in the gaps; finally, a DNA ligase seals the nicks. (B) The pJET plasmids containing the mutated versions of mutL were then digested with Xho I and Nco I, and (C) the mutated mutL inserts were then cloned into pOK12, (D) generating the plasmids pMC140, pMC141, pMC142, and pMC143 (see Supplementary Table 3 ). We then inserted the gentamicin resistance cassette with its own promoter (black arrow) downstream of mutL at the Nco I site. (E) Finally, we cloned the bb0212 gene downstream of the gent cassette to maintain the natural gene order in the B. burgdorferi chromosome. (F) Plasmids pMC148, pMC149, pMC150, and pMC151 were used to transform B. burgdorferi B31 wild-type clone 5A4 [20] and (G) GentR-KanS transformants were further analyzed for double recombination events.

    Techniques Used: Plasmid Preparation, Clone Assay

    8) Product Images from "Multiplex CRISPR-Cas9 mutagenesis of the phytochrome gene family in Physcomitrium (Physcomitrella) patens"

    Article Title: Multiplex CRISPR-Cas9 mutagenesis of the phytochrome gene family in Physcomitrium (Physcomitrella) patens

    Journal: Plant Molecular Biology

    doi: 10.1007/s11103-020-01103-x

    Identification of Cas9-mediated indels in high-resolution PAGE Short DNA fragments (~ 100 bp) surrounding each target site were amplified and separated on 15% polyacrylamide gels. a Lanes 1–5 and 6–11 show PHY5a and PHY2 products, respectively, from different lines amplified with Phusion High-Fidelity DNA Polymerase. Lanes 2 5 and 7, 10 11 show 2 and 1 bp deletions, respectively . b Lanes 12–17 and 18–22 show the same extracts amplified with Taq DNA Polymerase. Lanes 14 16 and 19 22 show 2 1 bp deletions, respectively
    Figure Legend Snippet: Identification of Cas9-mediated indels in high-resolution PAGE Short DNA fragments (~ 100 bp) surrounding each target site were amplified and separated on 15% polyacrylamide gels. a Lanes 1–5 and 6–11 show PHY5a and PHY2 products, respectively, from different lines amplified with Phusion High-Fidelity DNA Polymerase. Lanes 2 5 and 7, 10 11 show 2 and 1 bp deletions, respectively . b Lanes 12–17 and 18–22 show the same extracts amplified with Taq DNA Polymerase. Lanes 14 16 and 19 22 show 2 1 bp deletions, respectively

    Techniques Used: Polyacrylamide Gel Electrophoresis, Amplification

    9) Product Images from "Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells"

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122471

    Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.
    Figure Legend Snippet: Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.

    Techniques Used: Clone Assay, Amplification, Sequencing, Polymerase Chain Reaction, Variant Assay

    10) Product Images from "Cyclin A triggers Mitosis either via Greatwall or Cyclin B"

    Article Title: Cyclin A triggers Mitosis either via Greatwall or Cyclin B

    Journal: bioRxiv

    doi: 10.1101/501684

    Co-depletion of Ensa/ARPP19 or Greatwall and Cyclin B causes a prolonged prophase (A) Immuno-blot of B1 dd /B2 ko cells transfected with indicated Ctr or Ensa/ARPP19 siRNAs. Cells were blocked in Thymidine 12 hours after siRNA tranfection for 24 hours and released for 10 hours before sample preparation. Ensa/ARPP19 depletion is incomplete under these condition but causes a substantial decrease in pS67 phosphorylation indicating that PP2A:B55 is not inhibited. (B) As in (A) with Greatwall siRNA tranfetced cells. Note that DIA treatment causes a substantial change in Gwl phosphorylation but does not affect Ensa/ARPP19 S67 phosphorylation (see Panel A) indicating that Gwl is active in DIA treated cells, but that Cyclin B is required for Gwl phosphorylation at residues not required for activity. (C) Widefield Imaging. DIC (Grey), SiR-DNA (b/w), of Ensa/ARPP19 siRNA transfected DIA treated B1 dd /B2 ko cells. Time is indicated in hh:min. (D) Quantification of mitotic index of siRNA transfected P/A synchronised cells (See Figure 4B for representative images. (E) Intensity of anti-Lamin A/C pS22 antibody staining in siRNA transfected and DIA treated B1 dd /B2 ko cells. The swarm blots classify data from mitotic cells with intact (blue) or disassembled (red) Lamin A/C. The table above the panel indicates the median values and selected p-values are indicated (F) Same as in (E) for centrosome distance in mitotic cells (Data for (E and F) are from three repeats, n > 50 per repeat).
    Figure Legend Snippet: Co-depletion of Ensa/ARPP19 or Greatwall and Cyclin B causes a prolonged prophase (A) Immuno-blot of B1 dd /B2 ko cells transfected with indicated Ctr or Ensa/ARPP19 siRNAs. Cells were blocked in Thymidine 12 hours after siRNA tranfection for 24 hours and released for 10 hours before sample preparation. Ensa/ARPP19 depletion is incomplete under these condition but causes a substantial decrease in pS67 phosphorylation indicating that PP2A:B55 is not inhibited. (B) As in (A) with Greatwall siRNA tranfetced cells. Note that DIA treatment causes a substantial change in Gwl phosphorylation but does not affect Ensa/ARPP19 S67 phosphorylation (see Panel A) indicating that Gwl is active in DIA treated cells, but that Cyclin B is required for Gwl phosphorylation at residues not required for activity. (C) Widefield Imaging. DIC (Grey), SiR-DNA (b/w), of Ensa/ARPP19 siRNA transfected DIA treated B1 dd /B2 ko cells. Time is indicated in hh:min. (D) Quantification of mitotic index of siRNA transfected P/A synchronised cells (See Figure 4B for representative images. (E) Intensity of anti-Lamin A/C pS22 antibody staining in siRNA transfected and DIA treated B1 dd /B2 ko cells. The swarm blots classify data from mitotic cells with intact (blue) or disassembled (red) Lamin A/C. The table above the panel indicates the median values and selected p-values are indicated (F) Same as in (E) for centrosome distance in mitotic cells (Data for (E and F) are from three repeats, n > 50 per repeat).

    Techniques Used: Transfection, Sample Prep, Activity Assay, Imaging, Staining

    Mitotic progression in RPE-1 cells lacking Cyclins B1 and B2. Differential interference contrast and fluorescence widefield video microscopy of mock or DIA treated SiR-DNA labelled cells with indicated genotypes. Images show a progression of 6 hour time points (time inicated as hh:min).
    Figure Legend Snippet: Mitotic progression in RPE-1 cells lacking Cyclins B1 and B2. Differential interference contrast and fluorescence widefield video microscopy of mock or DIA treated SiR-DNA labelled cells with indicated genotypes. Images show a progression of 6 hour time points (time inicated as hh:min).

    Techniques Used: Fluorescence, Microscopy

    11) Product Images from "An Engineered T7 RNA Polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis"

    Article Title: An Engineered T7 RNA Polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis

    Journal: bioRxiv

    doi: 10.1101/2022.09.01.506264

    A flow diagram for an RNA polymerase fidelity assay. Polymerase fidelity was measured based on directly sequencing a large number of RT-PCR clones derived from mRNA transcribed from variant polymerases. A 1.7 kb firefly luciferase DNA template DNA was used with wild-type and variant T7 RNAPs to transcribe full-length mRNA transcripts. RNA was isolated and residual DNA was removed from the RNA samples by nuclease treatment. Samples were reverse-transcribed with Accuprime Reverse Transcriptase (Agilent) using an oligo-(dT) 25 primer annealing to the (A) tail on the luciferase template. The RT reaction was then PCR amplified using PHUSION ® high-fidelity DNA polymerase. Individual clones were picked and sequenced to > 20x coverage depth on the Ion Torrent PGM platform (Thermo Fisher) in multiplex. Reads were mapped against the expected sequenc, and mutations including small insertion, deletions, and single-nucleotide polymorphisms were observed.
    Figure Legend Snippet: A flow diagram for an RNA polymerase fidelity assay. Polymerase fidelity was measured based on directly sequencing a large number of RT-PCR clones derived from mRNA transcribed from variant polymerases. A 1.7 kb firefly luciferase DNA template DNA was used with wild-type and variant T7 RNAPs to transcribe full-length mRNA transcripts. RNA was isolated and residual DNA was removed from the RNA samples by nuclease treatment. Samples were reverse-transcribed with Accuprime Reverse Transcriptase (Agilent) using an oligo-(dT) 25 primer annealing to the (A) tail on the luciferase template. The RT reaction was then PCR amplified using PHUSION ® high-fidelity DNA polymerase. Individual clones were picked and sequenced to > 20x coverage depth on the Ion Torrent PGM platform (Thermo Fisher) in multiplex. Reads were mapped against the expected sequenc, and mutations including small insertion, deletions, and single-nucleotide polymorphisms were observed.

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Derivative Assay, Variant Assay, Luciferase, Isolation, Polymerase Chain Reaction, Amplification, Multiplex Assay

    12) Product Images from "Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction"

    Article Title: Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction

    Journal: Current Protocols in Microbiology

    doi: 10.1002/cpmc.89

    PCR to test the efficiency of polymerases in amplifying PCR products from supernatants from different spore concentrations of the A. fumigatus wild‐type strain with primers ITS1/D2 (expected PCR band sizes is ∼1.2 kb). ( A ) Phusion High‐Fidelity DNA polymerase (New England Biolabs). ( B ) MyTaq RED Mix DNA polymerase (Bioline). P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).
    Figure Legend Snippet: PCR to test the efficiency of polymerases in amplifying PCR products from supernatants from different spore concentrations of the A. fumigatus wild‐type strain with primers ITS1/D2 (expected PCR band sizes is ∼1.2 kb). ( A ) Phusion High‐Fidelity DNA polymerase (New England Biolabs). ( B ) MyTaq RED Mix DNA polymerase (Bioline). P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).

    Techniques Used: Polymerase Chain Reaction, Amplification, Negative Control

    13) Product Images from "Self Assembled Recombinant Proteins on Metallic Nanoparticles As Bimodal Imaging Probes"

    Article Title: Self Assembled Recombinant Proteins on Metallic Nanoparticles As Bimodal Imaging Probes

    Journal: JOM (Warrendale, Pa. : 1989)

    doi: 10.1007/s11837-018-03325-3

    Schematic representation of MBP-GFP-AuBP encoding genetic construct and DNA fragments amplified with Phusion High-fidelity DNA polymerase (NEB). Lane 1: O’RangeRuler 50 bp DNA Ladder (Thermo Scientific), lane 2: Negative control, lane 3: GFP-AuBP and GFP encoding DNA fragment.
    Figure Legend Snippet: Schematic representation of MBP-GFP-AuBP encoding genetic construct and DNA fragments amplified with Phusion High-fidelity DNA polymerase (NEB). Lane 1: O’RangeRuler 50 bp DNA Ladder (Thermo Scientific), lane 2: Negative control, lane 3: GFP-AuBP and GFP encoding DNA fragment.

    Techniques Used: Construct, Amplification, Negative Control

    14) Product Images from "Quantification of synthetic errors during chemical synthesis of DNA and its suppression by non-canonical nucleosides"

    Article Title: Quantification of synthetic errors during chemical synthesis of DNA and its suppression by non-canonical nucleosides

    Journal: Scientific Reports

    doi: 10.1038/s41598-022-16222-2

    Comparison of error rates derived from assembling reactions using different polymerases. The synthesized oligonucleotide in the same batch was split into three samples and prepared NGS library by using Q5 High-Fidelity DNA polymerase (Q5), Phusion High-Fidelity DNA polymerase (Phusion), or Takara Ex Taq (Ex). ( a ) Q5 vs Phusion. ( b ) Q5 vs Ex.
    Figure Legend Snippet: Comparison of error rates derived from assembling reactions using different polymerases. The synthesized oligonucleotide in the same batch was split into three samples and prepared NGS library by using Q5 High-Fidelity DNA polymerase (Q5), Phusion High-Fidelity DNA polymerase (Phusion), or Takara Ex Taq (Ex). ( a ) Q5 vs Phusion. ( b ) Q5 vs Ex.

    Techniques Used: Derivative Assay, Synthesized, Next-Generation Sequencing

    15) Product Images from "A temperature-dependent translational switch controls biofilm development in Vibrio cholerae"

    Article Title: A temperature-dependent translational switch controls biofilm development in Vibrio cholerae

    Journal: bioRxiv

    doi: 10.1101/2020.05.07.082040

    BipA protein levels are elevated at 22°C. A. Relative bipA expression in V. cholerae co969: bipA -flag or co969: hapR c: bipA -flag strains, carrying a chromosmal bipA -flag phusion, between rugose colonies grown at 37°C (37R) and smooth colonies grown at 22°C (22Sb), determined by qRT-PCR. Data are the average of 3 biological replicates, each containing 8 colonies pooled together. Error bars, standard deviation. B. Western blot showing the BipA-flag protein levels of co969: bipA -flag or co969: hapR c: bipA -flag rugose colonies grown at 37°C (37R) and smooth colonies grown at 22°C (22Sb). co969 and co969: hapR c strains were used as a negative controls, respectively. The gel is a representative of 5 independent experiments. The Coomassie staining of the membrane, used as control, is shown below. C. Sensitivity to partial digestion with trypsin of purified BipA-His pre-incubated at either 37°C or 22°C. D. Sensitivity to partial digestion with trypsin of BSA pre-incubated at either 37°C or 22°C. In both C and D , 25 μg aliquots of BipA-His or BSA, respectively, were pre-incubated at either 37°C or 22°C for 2 h; then, they were kept at room temperature for 5 min and subjected to digestion with 1.5 μg trypsin at room temperature during the incubated times: 0, 20 sec, 30 sec, 2 min and 5 min, for BipA-His; or 0, 10, 30, 45, 60 and 90 min, for BSA. The digested fragments were separated on a 10% acrylamide gel, and the gel was stained with Coomassie. E. Circular dichroism curve (mdeg represented against wavelength) of purified BipA-His pre-incubated at different temperatures (37°C, 22°C or 15°C) for 30 min, or pre-incubated at 37°C for 30 min and then sifted to 22°C and further incubated for 30 min.
    Figure Legend Snippet: BipA protein levels are elevated at 22°C. A. Relative bipA expression in V. cholerae co969: bipA -flag or co969: hapR c: bipA -flag strains, carrying a chromosmal bipA -flag phusion, between rugose colonies grown at 37°C (37R) and smooth colonies grown at 22°C (22Sb), determined by qRT-PCR. Data are the average of 3 biological replicates, each containing 8 colonies pooled together. Error bars, standard deviation. B. Western blot showing the BipA-flag protein levels of co969: bipA -flag or co969: hapR c: bipA -flag rugose colonies grown at 37°C (37R) and smooth colonies grown at 22°C (22Sb). co969 and co969: hapR c strains were used as a negative controls, respectively. The gel is a representative of 5 independent experiments. The Coomassie staining of the membrane, used as control, is shown below. C. Sensitivity to partial digestion with trypsin of purified BipA-His pre-incubated at either 37°C or 22°C. D. Sensitivity to partial digestion with trypsin of BSA pre-incubated at either 37°C or 22°C. In both C and D , 25 μg aliquots of BipA-His or BSA, respectively, were pre-incubated at either 37°C or 22°C for 2 h; then, they were kept at room temperature for 5 min and subjected to digestion with 1.5 μg trypsin at room temperature during the incubated times: 0, 20 sec, 30 sec, 2 min and 5 min, for BipA-His; or 0, 10, 30, 45, 60 and 90 min, for BSA. The digested fragments were separated on a 10% acrylamide gel, and the gel was stained with Coomassie. E. Circular dichroism curve (mdeg represented against wavelength) of purified BipA-His pre-incubated at different temperatures (37°C, 22°C or 15°C) for 30 min, or pre-incubated at 37°C for 30 min and then sifted to 22°C and further incubated for 30 min.

    Techniques Used: Expressing, Quantitative RT-PCR, Standard Deviation, Western Blot, Staining, Purification, Incubation, Acrylamide Gel Assay

    16) Product Images from "Global analysis of putative phospholipases in the malaria parasite Plasmodium falciparum reveals critical factors for parasite proliferation"

    Article Title: Global analysis of putative phospholipases in the malaria parasite Plasmodium falciparum reveals critical factors for parasite proliferation

    Journal: bioRxiv

    doi: 10.1101/2021.06.28.450158

    PNPLA2-KO parasites have a defect in the mtETC. A-G) Drug susceptibility assays of WT and PNPLA2-KO parasites using proguanil (A), atovaquone (B), myxothiazol (C), antimycin A (D), DSM1 (E), dihydroartemisinin (DHA, F), primaquine (G). Parasite growth was assessed by measuring DNA content using SYBR gold when exposed to varying concentrations of drugs for 96 h. The growth of DMSO-treated control parasites was set to 100%. Shown are means +/- SD of 3 to 6 independent experiments performed in duplicate. Calculated IC 50 values with 95% confidence intervals are shown below each graph. H) The artificial electron acceptor decylubiquinone (DCUQ) does not rescue growth of PNLA2-KO parasites. WT and PNPLA2-KO parasites were grown in presence of various concentrations of DCUQ for two parasites cycles and parasitemia was evaluated using flow cytometry. As positive control, WT parasites were additionally treated with 1,15 nM atovaquone. Shown are means +/- SD of three independent experiments. I) PNPLA2-KO parasites have a defect in sustaining normal ΔΨm. C2-arrested WT and PNPLA2-KO schizonts that had been treated with DMSO (solvent control), 200 nM or 1 µM of proguanil were stained with the mitochondrial potentiometric dye rhodamine123 (Rho123, green) and parasites with a strong, weak or absent mitochondrial rhodamine123 signal were quantified by fluorescence microscopy. Shown are means +/- SD of four independent experiments, in which a total of 352 to 414 schizonts were analyzed per cell line and condition. For statistical evaluation a one-way ANOVA followed by a Holm-Sidak multiple comparison test was performed (*p
    Figure Legend Snippet: PNPLA2-KO parasites have a defect in the mtETC. A-G) Drug susceptibility assays of WT and PNPLA2-KO parasites using proguanil (A), atovaquone (B), myxothiazol (C), antimycin A (D), DSM1 (E), dihydroartemisinin (DHA, F), primaquine (G). Parasite growth was assessed by measuring DNA content using SYBR gold when exposed to varying concentrations of drugs for 96 h. The growth of DMSO-treated control parasites was set to 100%. Shown are means +/- SD of 3 to 6 independent experiments performed in duplicate. Calculated IC 50 values with 95% confidence intervals are shown below each graph. H) The artificial electron acceptor decylubiquinone (DCUQ) does not rescue growth of PNLA2-KO parasites. WT and PNPLA2-KO parasites were grown in presence of various concentrations of DCUQ for two parasites cycles and parasitemia was evaluated using flow cytometry. As positive control, WT parasites were additionally treated with 1,15 nM atovaquone. Shown are means +/- SD of three independent experiments. I) PNPLA2-KO parasites have a defect in sustaining normal ΔΨm. C2-arrested WT and PNPLA2-KO schizonts that had been treated with DMSO (solvent control), 200 nM or 1 µM of proguanil were stained with the mitochondrial potentiometric dye rhodamine123 (Rho123, green) and parasites with a strong, weak or absent mitochondrial rhodamine123 signal were quantified by fluorescence microscopy. Shown are means +/- SD of four independent experiments, in which a total of 352 to 414 schizonts were analyzed per cell line and condition. For statistical evaluation a one-way ANOVA followed by a Holm-Sidak multiple comparison test was performed (*p

    Techniques Used: Flow Cytometry, Positive Control, Staining, Fluorescence, Microscopy

    17) Product Images from "Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B"

    Article Title: Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B

    Journal: The EMBO Journal

    doi: 10.15252/embj.2020104419

    Mitotic phenotypes of DIA ‐treated B1dd/B2ko cells Stills from live‐cell imaging of asynchronously dividing B1 dd /B2 ko cells (DIC (top) and SiR‐DNA (bottom), time is indicated as hh:min, and scale bar indicates 20 μm). A successful cell division in untreated B1 dd /B2 ko cells. DIA‐treated B1 dd /B2 ko cells that fail to segregate sister chromatids and initiate cell division. DIA‐treated B1 dd /B2 ko cells that attempt segregation and cytokinesis but fail to separate resulting in binuclear cells. DIA‐treated B1 dd /B2 ko cells that fail to undergo NEBD and exit mitosis from prophase. All cells were imaged four to 6 h after DIA treatment.
    Figure Legend Snippet: Mitotic phenotypes of DIA ‐treated B1dd/B2ko cells Stills from live‐cell imaging of asynchronously dividing B1 dd /B2 ko cells (DIC (top) and SiR‐DNA (bottom), time is indicated as hh:min, and scale bar indicates 20 μm). A successful cell division in untreated B1 dd /B2 ko cells. DIA‐treated B1 dd /B2 ko cells that fail to segregate sister chromatids and initiate cell division. DIA‐treated B1 dd /B2 ko cells that attempt segregation and cytokinesis but fail to separate resulting in binuclear cells. DIA‐treated B1 dd /B2 ko cells that fail to undergo NEBD and exit mitosis from prophase. All cells were imaged four to 6 h after DIA treatment.

    Techniques Used: Live Cell Imaging

    Cyclin A2 is essential to trigger mitotic entry in G2 phase Time‐lapse images of PCNA‐mRuby‐tagged A2 dd cells. The imaging sequence was started at the time of doxycycline addition, and degron activity was triggered 3 h later by addition of IAA and Asv or PBS (indicated by dashed line). Time is shown as hh:min; scale bar equals 10 μm. For further analysis, cells were chosen that had dissipated their PCNA foci before the addition of DIA to ensure that cyclin A2 degradation was triggered in G2 phase. Single‐cell analysis of 40 cells per condition treated as described in (A); each line represents the timing of G2/M/G1 progression for a single cell. A2 dd cells were pulsed with EdU to label S phase then protein degradation was induced by DIA addition for 5 h and before fixation and Edu/CENPF/DAPI staining. Cells that were in G2 at the time of DIA or mock treatment were identified as CENPF‐positive, EdU‐negative. Mitotic cells were excluded based on nuclei morphology. The bar plots show mean percentage of EdU‐negative/CENPF‐positive cells of three repeats; error bars indicate standard deviation. Images from time‐lapse sequence of mitosis showing cell division in controls and after addition of 0.5 μM PD‐166285 in DIA‐treated A2 dd labelled with SiR‐DNA (time is indicated as hh:min; the scale bars represent 20 μm). Accumulative index of mitotic entry measured by time‐lapse microscopy in A2 dd cells following mock or DIA treatment and addition of Wee1 inhibitors MK1775 (1 μM) or PD‐166285 (0.5 μM). Asynchronous cells were DIA‐treated for 4 h and then imaged for 6 h in 5‐min intervals. (Data from three repeats, n > 500 cells per condition; standard deviation is indicated by shaded area). Quantification of metaphase duration in A2 dd cells treated with DIA and Wee1 inhibitors as indicated ( n > 50 per repeat, the boxplot indicates median, first and third quartile and minimum/maximum values.) A2 dd cells were transfected with Ctr or B55α/δ siRNA. Forty‐eight hours after transfection, the cells were treated with Thymidine for 24 h and then released. Ten hours later, the cells were probed for B55 depletion by immunoblotting using a pan‐B55 monoclonal antibody. Lack of B55 partially rescues mitotic entry in DIA‐treated A2 dd cells. Cells transfected with Ctr siRNA or B55α/δ siRNA as in (B) were released from Thymidine, mock‐ or DIA‐treated and analysed by widefield live‐cell imaging 8 h after the release. The cells were imaged every 10 min for 15 h and manually scored for mitotic index. The lines represent mean values of three independent experiments, and standard deviation is indicated by the coloured areas ( n > 100 per experiment).
    Figure Legend Snippet: Cyclin A2 is essential to trigger mitotic entry in G2 phase Time‐lapse images of PCNA‐mRuby‐tagged A2 dd cells. The imaging sequence was started at the time of doxycycline addition, and degron activity was triggered 3 h later by addition of IAA and Asv or PBS (indicated by dashed line). Time is shown as hh:min; scale bar equals 10 μm. For further analysis, cells were chosen that had dissipated their PCNA foci before the addition of DIA to ensure that cyclin A2 degradation was triggered in G2 phase. Single‐cell analysis of 40 cells per condition treated as described in (A); each line represents the timing of G2/M/G1 progression for a single cell. A2 dd cells were pulsed with EdU to label S phase then protein degradation was induced by DIA addition for 5 h and before fixation and Edu/CENPF/DAPI staining. Cells that were in G2 at the time of DIA or mock treatment were identified as CENPF‐positive, EdU‐negative. Mitotic cells were excluded based on nuclei morphology. The bar plots show mean percentage of EdU‐negative/CENPF‐positive cells of three repeats; error bars indicate standard deviation. Images from time‐lapse sequence of mitosis showing cell division in controls and after addition of 0.5 μM PD‐166285 in DIA‐treated A2 dd labelled with SiR‐DNA (time is indicated as hh:min; the scale bars represent 20 μm). Accumulative index of mitotic entry measured by time‐lapse microscopy in A2 dd cells following mock or DIA treatment and addition of Wee1 inhibitors MK1775 (1 μM) or PD‐166285 (0.5 μM). Asynchronous cells were DIA‐treated for 4 h and then imaged for 6 h in 5‐min intervals. (Data from three repeats, n > 500 cells per condition; standard deviation is indicated by shaded area). Quantification of metaphase duration in A2 dd cells treated with DIA and Wee1 inhibitors as indicated ( n > 50 per repeat, the boxplot indicates median, first and third quartile and minimum/maximum values.) A2 dd cells were transfected with Ctr or B55α/δ siRNA. Forty‐eight hours after transfection, the cells were treated with Thymidine for 24 h and then released. Ten hours later, the cells were probed for B55 depletion by immunoblotting using a pan‐B55 monoclonal antibody. Lack of B55 partially rescues mitotic entry in DIA‐treated A2 dd cells. Cells transfected with Ctr siRNA or B55α/δ siRNA as in (B) were released from Thymidine, mock‐ or DIA‐treated and analysed by widefield live‐cell imaging 8 h after the release. The cells were imaged every 10 min for 15 h and manually scored for mitotic index. The lines represent mean values of three independent experiments, and standard deviation is indicated by the coloured areas ( n > 100 per experiment).

    Techniques Used: Imaging, Sequencing, Activity Assay, Single-cell Analysis, Staining, Standard Deviation, Time-lapse Microscopy, Transfection, Live Cell Imaging

    Mitotic progression in RPE ‐1 cells lacking cyclins B1 and B2 Differential interference contrast (DIC) and fluorescence widefield video microscopy of mock or DIA‐treated SiR‐DNA‐labelled cells with indicated genotypes. Images show a progression of 6 h time points (time indicated as hh:min, scale bar indicates 20 μm, and arrows point to dividing cells).
    Figure Legend Snippet: Mitotic progression in RPE ‐1 cells lacking cyclins B1 and B2 Differential interference contrast (DIC) and fluorescence widefield video microscopy of mock or DIA‐treated SiR‐DNA‐labelled cells with indicated genotypes. Images show a progression of 6 h time points (time indicated as hh:min, scale bar indicates 20 μm, and arrows point to dividing cells).

    Techniques Used: Fluorescence, Microscopy

    Greatwall and cyclin B synergise in triggering NEBD but not prophase Representative images from live‐cell imaging of siRNA‐transfected B1 dd /B2 ko cells (FusionRed‐H2B in red, SiR‐Tubulin in white, scale bar = 10 μm). Bar plot panels on the right show single‐cell analysis of 10 cells manually scored for length mitosis pre‐NEBD and post‐NEBD. Entry into prophase was scored by cell rounding and NEBD was identified by influx of Tubulin in the nucleus. Widefield imaging. DIC (Grey), SiR‐DNA (b/w), of ENSA/ARPP19 siRNA‐transfected DIA‐treated B1 dd /B2 ko cells. Time is indicated in hh:min, scale bar = 10 μm. Panels of representative immunofluorescence images of siRNA‐transfected and P/A‐treated cells treated as indicated, scale bar = 10 μm. Quantification of mitotic index of siRNA‐transfected P/A synchronised cells (See Fig. 4 B for representative images; mean value of three repeats, n > 500 per repeat; error bars indicate standard deviation). Quantification of mitotic B1 dd /B2 ko cells following siRNA transfection to deplete Greatwall (siGwl) or ENSA/ARPP19 (siE/A) and/or DIA treatment. Frequencies of mitotic cells with intact (blue) and disassembled (red) Lamin A/C staining are plotted (mean value of three repeats, n > 50 per repeat; error bars indicate standard deviation). As in (E) quantification of control‐ and DIA‐treated mitotic B1 dd /B2 ko cells following siRNA transfection to deplete Greatwall and B55α/δ. Frequencies of mitotic cells with intact (blue) and disassembled (red) Lamin A/C staining are plotted (mean value of three repeats, n > 50 per repeat; error bars indicate standard deviation). Intensity of pSP‐antibody staining in siRNA‐transfected and DIA‐treated B1 dd /B2 ko cells. The swarm‐blots classify data from mitotic cells with intact (blue) or disassembled (red) Lamin A/C. As in (G) intensity of anti‐Lamin A/C pS22 antibody staining in siRNA‐transfected and DIA‐treated B1 dd /B2 ko cells. Same as in (G) and (H), data for centrosome distance in mitotic cells. Data for G‐I are from three repeats, n > 50 per repeat; the bars indicate median, first and third quartile as well as minimum and maximum values; data are from three repeats, n = 50 cells per repeat).
    Figure Legend Snippet: Greatwall and cyclin B synergise in triggering NEBD but not prophase Representative images from live‐cell imaging of siRNA‐transfected B1 dd /B2 ko cells (FusionRed‐H2B in red, SiR‐Tubulin in white, scale bar = 10 μm). Bar plot panels on the right show single‐cell analysis of 10 cells manually scored for length mitosis pre‐NEBD and post‐NEBD. Entry into prophase was scored by cell rounding and NEBD was identified by influx of Tubulin in the nucleus. Widefield imaging. DIC (Grey), SiR‐DNA (b/w), of ENSA/ARPP19 siRNA‐transfected DIA‐treated B1 dd /B2 ko cells. Time is indicated in hh:min, scale bar = 10 μm. Panels of representative immunofluorescence images of siRNA‐transfected and P/A‐treated cells treated as indicated, scale bar = 10 μm. Quantification of mitotic index of siRNA‐transfected P/A synchronised cells (See Fig. 4 B for representative images; mean value of three repeats, n > 500 per repeat; error bars indicate standard deviation). Quantification of mitotic B1 dd /B2 ko cells following siRNA transfection to deplete Greatwall (siGwl) or ENSA/ARPP19 (siE/A) and/or DIA treatment. Frequencies of mitotic cells with intact (blue) and disassembled (red) Lamin A/C staining are plotted (mean value of three repeats, n > 50 per repeat; error bars indicate standard deviation). As in (E) quantification of control‐ and DIA‐treated mitotic B1 dd /B2 ko cells following siRNA transfection to deplete Greatwall and B55α/δ. Frequencies of mitotic cells with intact (blue) and disassembled (red) Lamin A/C staining are plotted (mean value of three repeats, n > 50 per repeat; error bars indicate standard deviation). Intensity of pSP‐antibody staining in siRNA‐transfected and DIA‐treated B1 dd /B2 ko cells. The swarm‐blots classify data from mitotic cells with intact (blue) or disassembled (red) Lamin A/C. As in (G) intensity of anti‐Lamin A/C pS22 antibody staining in siRNA‐transfected and DIA‐treated B1 dd /B2 ko cells. Same as in (G) and (H), data for centrosome distance in mitotic cells. Data for G‐I are from three repeats, n > 50 per repeat; the bars indicate median, first and third quartile as well as minimum and maximum values; data are from three repeats, n = 50 cells per repeat).

    Techniques Used: Live Cell Imaging, Transfection, Single-cell Analysis, Imaging, Immunofluorescence, Standard Deviation, Staining

    Cell proliferation in the absence of cyclin A2 and B‐type cyclins Cyclin B2 knock‐out and induced degradation by immunoblotting. Indicated cell lines were analysed 24 h after mock or Dox/IAA/Asv (DIA) treatment using the indicated antibodies to confirm homozygous gene tagging and efficiency of protein degradation. Cell cycle analysis 24 h after DIA treatment. Cells were analysed by EdU labelling, PI staining and FACS analysis. The histograms show the PI intensities while the dot plots show EdU incorporation ( y ‐axis) vs PI intensity ( x ‐axis). Gating of cell cycle phases is indicated by colour (G1—black, S phase—green, G2/M phase—orange) Cell cycle phase frequencies quantified from flow cytometry data (shown in (B)) in indicated cell lines 24 h after DIA treatment, ( n = 3 experiments, s.d. indicated by error bars). Cell proliferation of A2 dd , CycB1 dd and B1 dd /B2 ko following mock or DIA treatment. One thousand cells were plated in each well (diameter 3.5 cm) and incubated for 10 days before methanol fixation and Crystal Violet staining. Kinetics of mitotic entry as measured by time‐lapse microscopy in A2 dd and B1 dd /B2 ko cells following mock or 4‐h DIA treatment of asynchronous cells. The cells were imaged for 16 h with 5‐min intervals using widefield DIC; mitotic entry was manually scored by detecting cell rounding. Curves display the cumulative mitotic index (data from three repeats, n > 500 cells per condition, s.d. indicated by shaded area). qPCR analysis of cyclin B3 mRNA levels, following 72‐h depletion in B1 dd /B2 ko cells. For quantification, we used primers to amplify two control mRNAs, TATA‐binding protein and actin. The plot shows the levels of CycB3 siRNA‐depleted mRNA relative to Ctr siRNA‐transfected cells. (Bars indicate the mean of three independent experiments; error bars indicate the standard deviation between these three repeats.) Mitotic index measurements of B1 dd /B2 ko cells with the indicated treatments. Cells were transfected with siRNA for 72 h; after 36 h, they were blocked for 24 h with Thymidine and fixed 12 h after release from Thymidine. ProTAME and Apcin were added for the final 2 h before fixation. Mitotic cells were scored based on DAPI staining and on condensed chromosome formation. The bar plots show mean values of three biological repeats ( n = 100 per repeat and sample, error bars indicate standard deviation, and P ‐values were calculated using an independent two‐sample t‐test). Cyclin A2 siRNA depletion in MCF7 and MCF10A cells. The cells were transfected with Ctr or cyclin A2 siRNA for indicated length of time and probed for cyclin A2 levels by immunoblotting. Cyclin A2 siRNA depletion causes endoreplication. Following 72 h of siRNA transfection, MCF7 and MCF10A cells were analysed by PI staining and FACS. The histograms show the changes in DNA content (PI Int.) towards > 4N following cyclin A2 depletion. Cyclin A2 siRNA and degron depletion in RPE‐1 cells. RPE‐1 OsTIR1 and RPE‐1 A2 dd cells were subjected to 72 h of cyclin A2 siRNA depletion and/or of DIA treatment as indicated and probed for cyclin A2 levels by immunoblotting. The longer exposure (L.E.) reveals incomplete depletion of cyclin 2 by siRNA. Cyclin A degron depletion causes accumulation of cells in G2 phase. Following 72 h of siRNA transfection or DIA treatment, RPE‐1 OsTIR1 and A2 dd cells were analysed by PI staining and FACS. The histograms show the changes in DNA content (PI Int.) towards > 4N following cyclin siRNA A2 depletion in RPE‐1 OsTIR1 cells, while DIA treatment in A2 dd cells does cause an increase in the 4N but not > 4N peak.
    Figure Legend Snippet: Cell proliferation in the absence of cyclin A2 and B‐type cyclins Cyclin B2 knock‐out and induced degradation by immunoblotting. Indicated cell lines were analysed 24 h after mock or Dox/IAA/Asv (DIA) treatment using the indicated antibodies to confirm homozygous gene tagging and efficiency of protein degradation. Cell cycle analysis 24 h after DIA treatment. Cells were analysed by EdU labelling, PI staining and FACS analysis. The histograms show the PI intensities while the dot plots show EdU incorporation ( y ‐axis) vs PI intensity ( x ‐axis). Gating of cell cycle phases is indicated by colour (G1—black, S phase—green, G2/M phase—orange) Cell cycle phase frequencies quantified from flow cytometry data (shown in (B)) in indicated cell lines 24 h after DIA treatment, ( n = 3 experiments, s.d. indicated by error bars). Cell proliferation of A2 dd , CycB1 dd and B1 dd /B2 ko following mock or DIA treatment. One thousand cells were plated in each well (diameter 3.5 cm) and incubated for 10 days before methanol fixation and Crystal Violet staining. Kinetics of mitotic entry as measured by time‐lapse microscopy in A2 dd and B1 dd /B2 ko cells following mock or 4‐h DIA treatment of asynchronous cells. The cells were imaged for 16 h with 5‐min intervals using widefield DIC; mitotic entry was manually scored by detecting cell rounding. Curves display the cumulative mitotic index (data from three repeats, n > 500 cells per condition, s.d. indicated by shaded area). qPCR analysis of cyclin B3 mRNA levels, following 72‐h depletion in B1 dd /B2 ko cells. For quantification, we used primers to amplify two control mRNAs, TATA‐binding protein and actin. The plot shows the levels of CycB3 siRNA‐depleted mRNA relative to Ctr siRNA‐transfected cells. (Bars indicate the mean of three independent experiments; error bars indicate the standard deviation between these three repeats.) Mitotic index measurements of B1 dd /B2 ko cells with the indicated treatments. Cells were transfected with siRNA for 72 h; after 36 h, they were blocked for 24 h with Thymidine and fixed 12 h after release from Thymidine. ProTAME and Apcin were added for the final 2 h before fixation. Mitotic cells were scored based on DAPI staining and on condensed chromosome formation. The bar plots show mean values of three biological repeats ( n = 100 per repeat and sample, error bars indicate standard deviation, and P ‐values were calculated using an independent two‐sample t‐test). Cyclin A2 siRNA depletion in MCF7 and MCF10A cells. The cells were transfected with Ctr or cyclin A2 siRNA for indicated length of time and probed for cyclin A2 levels by immunoblotting. Cyclin A2 siRNA depletion causes endoreplication. Following 72 h of siRNA transfection, MCF7 and MCF10A cells were analysed by PI staining and FACS. The histograms show the changes in DNA content (PI Int.) towards > 4N following cyclin A2 depletion. Cyclin A2 siRNA and degron depletion in RPE‐1 cells. RPE‐1 OsTIR1 and RPE‐1 A2 dd cells were subjected to 72 h of cyclin A2 siRNA depletion and/or of DIA treatment as indicated and probed for cyclin A2 levels by immunoblotting. The longer exposure (L.E.) reveals incomplete depletion of cyclin 2 by siRNA. Cyclin A degron depletion causes accumulation of cells in G2 phase. Following 72 h of siRNA transfection or DIA treatment, RPE‐1 OsTIR1 and A2 dd cells were analysed by PI staining and FACS. The histograms show the changes in DNA content (PI Int.) towards > 4N following cyclin siRNA A2 depletion in RPE‐1 OsTIR1 cells, while DIA treatment in A2 dd cells does cause an increase in the 4N but not > 4N peak.

    Techniques Used: Knock-Out, Cell Cycle Assay, Staining, FACS, Flow Cytometry, Incubation, Time-lapse Microscopy, Real-time Polymerase Chain Reaction, Binding Assay, Transfection, Standard Deviation

    Rescue of cyclin B phenotypes by induced expression of cyclin B B1 dd /B2 ko cells containing indicated inducible constructs were released from a single Thymidine arrest and treated with DIA to deplete endogenous cyclin B1 and induce expression of YFP, CycB1‐YFP (B1‐WT) or CycB1‐YFP‐NLS (B1‐NLS). Imaging (SiR‐DNA and YFP) was initiated 10 h after the release. The images displayed are from the time‐lapse sequence at the indicated time points in hh:min. The scale bar represents a length of 10 μm. B1‐WT‐ and B1‐NLS‐inducible cell lines were analysed for DIA‐induced cyclin B1 induction/depletion. Samples were collected at indicated time points and probed by immunoblotting with cyclin B1 and α‐tubulin antibodies (* indicates non‐specific band). Quantification of the DIA‐induced cell division phenotypes following induction of YFP, CycB1‐YFP and CycB1‐YFP‐NLS induction. Data were collected from three independent experiments ( n = 50), and the error bars indicate standard deviation between the three data sets.
    Figure Legend Snippet: Rescue of cyclin B phenotypes by induced expression of cyclin B B1 dd /B2 ko cells containing indicated inducible constructs were released from a single Thymidine arrest and treated with DIA to deplete endogenous cyclin B1 and induce expression of YFP, CycB1‐YFP (B1‐WT) or CycB1‐YFP‐NLS (B1‐NLS). Imaging (SiR‐DNA and YFP) was initiated 10 h after the release. The images displayed are from the time‐lapse sequence at the indicated time points in hh:min. The scale bar represents a length of 10 μm. B1‐WT‐ and B1‐NLS‐inducible cell lines were analysed for DIA‐induced cyclin B1 induction/depletion. Samples were collected at indicated time points and probed by immunoblotting with cyclin B1 and α‐tubulin antibodies (* indicates non‐specific band). Quantification of the DIA‐induced cell division phenotypes following induction of YFP, CycB1‐YFP and CycB1‐YFP‐NLS induction. Data were collected from three independent experiments ( n = 50), and the error bars indicate standard deviation between the three data sets.

    Techniques Used: Expressing, Construct, Imaging, Sequencing, Standard Deviation

    18) Product Images from "Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction"

    Article Title: Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction

    Journal: Current Protocols in Microbiology

    doi: 10.1002/cpmc.89

    PCR to test the efficiency of polymerases in amplifying PCR products from supernatants from different spore concentrations of the A. fumigatus wild‐type strain with primers ITS1/D2 (expected PCR band sizes is ∼1.2 kb). ( A ) Phusion High‐Fidelity DNA polymerase (New England Biolabs). ( B ) MyTaq RED Mix DNA polymerase (Bioline). P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).
    Figure Legend Snippet: PCR to test the efficiency of polymerases in amplifying PCR products from supernatants from different spore concentrations of the A. fumigatus wild‐type strain with primers ITS1/D2 (expected PCR band sizes is ∼1.2 kb). ( A ) Phusion High‐Fidelity DNA polymerase (New England Biolabs). ( B ) MyTaq RED Mix DNA polymerase (Bioline). P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).

    Techniques Used: Polymerase Chain Reaction, Amplification, Negative Control

    19) Product Images from "Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost"

    Article Title: Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0153158

    “Dissection” of the Gibson assembly into its component reactions. In the Gibson assembly, single-stranded 3’-overhangs are produced using T5 exonuclease. After insert-to-plasmid annealing at complementary single-stranded DNA ends, gaps are filled-in by Phusion DNA polymerase, and finally, Taq DNA ligase ligates the nicks. Here, we compared the efficiencies at which insert-plasmid mixtures transformed chemically competent E . coli cells. We used untreated insert-plasmid mixtures (co-transformation cloning), insert-plasmid mixtures treated with T5 exonuclease (sequence- and ligation-independent cloning), insert-plasmid mixtures treated with T5 exonuclease and Phusion DNA polymerase (sequence- and ligation-independent cloning plus gap filling), and insert-plasmid mixtures treated with T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase (Gibson assembly). A) Data points represent the number of positive (blue) colonies averaged over three experiments ±SD. B) Data points represent the percentage of positive colonies averaged over three experiments ±SD.
    Figure Legend Snippet: “Dissection” of the Gibson assembly into its component reactions. In the Gibson assembly, single-stranded 3’-overhangs are produced using T5 exonuclease. After insert-to-plasmid annealing at complementary single-stranded DNA ends, gaps are filled-in by Phusion DNA polymerase, and finally, Taq DNA ligase ligates the nicks. Here, we compared the efficiencies at which insert-plasmid mixtures transformed chemically competent E . coli cells. We used untreated insert-plasmid mixtures (co-transformation cloning), insert-plasmid mixtures treated with T5 exonuclease (sequence- and ligation-independent cloning), insert-plasmid mixtures treated with T5 exonuclease and Phusion DNA polymerase (sequence- and ligation-independent cloning plus gap filling), and insert-plasmid mixtures treated with T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase (Gibson assembly). A) Data points represent the number of positive (blue) colonies averaged over three experiments ±SD. B) Data points represent the percentage of positive colonies averaged over three experiments ±SD.

    Techniques Used: Produced, Plasmid Preparation, Transformation Assay, Clone Assay, Sequencing, Ligation

    20) Product Images from "Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template"

    Article Title: Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template

    Journal: Synthetic Biology

    doi: 10.1093/synbio/ysaa030

    Schematic overview of the SLUPT strategy. Step 1: The gene of interest is amplified with a 5′ phosphorylated top strand primer and dNTP’s containing dU (blue). The primer for the bottom strand is not phosphorylated. Optional, nonhomologous regions (e.g. to introduce restriction enzyme sites) are shown in green. Step 2: The phosphorylated strand is selectively degraded by lambda exonuclease to create the uracil-containing single stranded template. Step 3: An end-primer complementary to the 3′ terminus and 5′ phosphorylated internal primers containing altered bases are annealed to the uracil containing single strand template. Altered bases depicted as X’s in red box. Gap filling and ligation are performed by Phusion-U and Taq ligase to create a mutated, complementary strand. Step 4: The Uracil-containing single stranded template is digested by UDG. Step 5: The single-stranded product is made double stranded and amplified by PCR.
    Figure Legend Snippet: Schematic overview of the SLUPT strategy. Step 1: The gene of interest is amplified with a 5′ phosphorylated top strand primer and dNTP’s containing dU (blue). The primer for the bottom strand is not phosphorylated. Optional, nonhomologous regions (e.g. to introduce restriction enzyme sites) are shown in green. Step 2: The phosphorylated strand is selectively degraded by lambda exonuclease to create the uracil-containing single stranded template. Step 3: An end-primer complementary to the 3′ terminus and 5′ phosphorylated internal primers containing altered bases are annealed to the uracil containing single strand template. Altered bases depicted as X’s in red box. Gap filling and ligation are performed by Phusion-U and Taq ligase to create a mutated, complementary strand. Step 4: The Uracil-containing single stranded template is digested by UDG. Step 5: The single-stranded product is made double stranded and amplified by PCR.

    Techniques Used: Amplification, Introduce, Ligation, Polymerase Chain Reaction

    21) Product Images from "Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction"

    Article Title: Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction

    Journal: Current Protocols in Microbiology

    doi: 10.1002/cpmc.89

    PCR to test the efficiency of polymerases in amplifying PCR products from supernatants from different spore concentrations of the A. fumigatus wild‐type strain with primers ITS1/D2 (expected PCR band sizes is ∼1.2 kb). ( A ) Phusion High‐Fidelity DNA polymerase (New England Biolabs). ( B ) MyTaq RED Mix DNA polymerase (Bioline). P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).
    Figure Legend Snippet: PCR to test the efficiency of polymerases in amplifying PCR products from supernatants from different spore concentrations of the A. fumigatus wild‐type strain with primers ITS1/D2 (expected PCR band sizes is ∼1.2 kb). ( A ) Phusion High‐Fidelity DNA polymerase (New England Biolabs). ( B ) MyTaq RED Mix DNA polymerase (Bioline). P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).

    Techniques Used: Polymerase Chain Reaction, Amplification, Negative Control

    22) Product Images from "A comprehensive assay for targeted multiplex amplification of human DNA sequences"

    Article Title: A comprehensive assay for targeted multiplex amplification of human DNA sequences

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0803240105

    The median CEL intensities for each amplicon obtained by using Stoffel DNA polymerase and Phusion DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were
    Figure Legend Snippet: The median CEL intensities for each amplicon obtained by using Stoffel DNA polymerase and Phusion DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were

    Techniques Used: Amplification

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    New England Biolabs phusion high fidelity dna polymerase
    Cloned pre-mir-122 stem-loop region sequences from HepG2 <t>DNA</t> show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with <t>Phusion</t> high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs pcr amplified
    Sulfadiazine foliar spray selection of T 1 pCS4-GFP transgenic plants grown at a high density in soil . Sulfadiazine solution (500 mg/L with 0.03% L-77 silwet) was applied every third day for a total of three applications. A) Plant growth 21 days after the initial sulfadiazine application. B) Schematic representation of the CS4-GFP <t>T-DNA,</t> where the dashed line represents the region <t>PCR</t> amplified with sul I primers. C) PCR amplification results confirming the presence of sul I in 17 randomly chosen sulfadiazine resistant plants. The dash (-) indicates a water negative control, and the plus (+) indicates a plasmid positive control. A size standard labeled in kilobase pairs (kb) is shown.
    Pcr Amplified, 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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    Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.

    Journal: PLoS ONE

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells

    doi: 10.1371/journal.pone.0122471

    Figure Lengend Snippet: Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.

    Article Snippet: The observed error rate was ~14-fold higher than that reported for Phusion High-Fidelity DNA Polymerase (GC Buffer) (New England Biolabs; ) which, if this error rate is correct, suggests that these changes are unlikely to have arisen solely as a result of PCR errors.

    Techniques: Clone Assay, Amplification, Sequencing, Polymerase Chain Reaction, Variant Assay

    Olig2.5 inhibited archaeal family B DNA polymerases. An E. coli DH5α colony harbouring the 2.6 kb upstream sequence of Olig2 in pGL3-Basic vector was cultured and 1 μl of the culture was directly used as template for PCR. Olig2F and OligTSSR were used as primers to amplify the 2 kb fragment. Olig2.5 was added to see its inhibition to various DNAP. DNAP: DNA polymerase. LA: LA Taq DNAP. Q5: Q5 High-Fidelity DNAP. PS: PrimeSTAR HS DNAP. GXL: PrimeSTAR GXL DNAP. Phusion: Phusion High-Fidelity DNAP. Cobuddy: Cobuddy Super Fidelity DNAP. KOD: KOD DNAP.

    Journal: Scientific Reports

    Article Title: TT(N)mGCCTC inhibits archaeal family B DNA polymerases

    doi: 10.1038/s41598-018-20127-4

    Figure Lengend Snippet: Olig2.5 inhibited archaeal family B DNA polymerases. An E. coli DH5α colony harbouring the 2.6 kb upstream sequence of Olig2 in pGL3-Basic vector was cultured and 1 μl of the culture was directly used as template for PCR. Olig2F and OligTSSR were used as primers to amplify the 2 kb fragment. Olig2.5 was added to see its inhibition to various DNAP. DNAP: DNA polymerase. LA: LA Taq DNAP. Q5: Q5 High-Fidelity DNAP. PS: PrimeSTAR HS DNAP. GXL: PrimeSTAR GXL DNAP. Phusion: Phusion High-Fidelity DNAP. Cobuddy: Cobuddy Super Fidelity DNAP. KOD: KOD DNAP.

    Article Snippet: Phusion® High-Fidelity DNA Polymerase (Phusion) and Q5® High-Fidelity DNA Polymerase (Q5) were from New England Biolabs.

    Techniques: Sequencing, Plasmid Preparation, Cell Culture, Polymerase Chain Reaction, Inhibition

    PNPLA2-KO parasites have a defect in the mtETC. A-G) Drug susceptibility assays of WT and PNPLA2-KO parasites using proguanil (A), atovaquone (B), myxothiazol (C), antimycin A (D), DSM1 (E), dihydroartemisinin (DHA, F), primaquine (G). Parasite growth was assessed by measuring DNA content using SYBR gold when exposed to varying concentrations of drugs for 96 h. The growth of DMSO-treated control parasites was set to 100%. Shown are means +/- SD of 3 to 6 independent experiments performed in duplicate. Calculated IC 50 values with 95% confidence intervals are shown below each graph. H) The artificial electron acceptor decylubiquinone (DCUQ) does not rescue growth of PNLA2-KO parasites. WT and PNPLA2-KO parasites were grown in presence of various concentrations of DCUQ for two parasites cycles and parasitemia was evaluated using flow cytometry. As positive control, WT parasites were additionally treated with 1,15 nM atovaquone. Shown are means +/- SD of three independent experiments. I) PNPLA2-KO parasites have a defect in sustaining normal ΔΨm. C2-arrested WT and PNPLA2-KO schizonts that had been treated with DMSO (solvent control), 200 nM or 1 µM of proguanil were stained with the mitochondrial potentiometric dye rhodamine123 (Rho123, green) and parasites with a strong, weak or absent mitochondrial rhodamine123 signal were quantified by fluorescence microscopy. Shown are means +/- SD of four independent experiments, in which a total of 352 to 414 schizonts were analyzed per cell line and condition. For statistical evaluation a one-way ANOVA followed by a Holm-Sidak multiple comparison test was performed (*p

    Journal: bioRxiv

    Article Title: Global analysis of putative phospholipases in the malaria parasite Plasmodium falciparum reveals critical factors for parasite proliferation

    doi: 10.1101/2021.06.28.450158

    Figure Lengend Snippet: PNPLA2-KO parasites have a defect in the mtETC. A-G) Drug susceptibility assays of WT and PNPLA2-KO parasites using proguanil (A), atovaquone (B), myxothiazol (C), antimycin A (D), DSM1 (E), dihydroartemisinin (DHA, F), primaquine (G). Parasite growth was assessed by measuring DNA content using SYBR gold when exposed to varying concentrations of drugs for 96 h. The growth of DMSO-treated control parasites was set to 100%. Shown are means +/- SD of 3 to 6 independent experiments performed in duplicate. Calculated IC 50 values with 95% confidence intervals are shown below each graph. H) The artificial electron acceptor decylubiquinone (DCUQ) does not rescue growth of PNLA2-KO parasites. WT and PNPLA2-KO parasites were grown in presence of various concentrations of DCUQ for two parasites cycles and parasitemia was evaluated using flow cytometry. As positive control, WT parasites were additionally treated with 1,15 nM atovaquone. Shown are means +/- SD of three independent experiments. I) PNPLA2-KO parasites have a defect in sustaining normal ΔΨm. C2-arrested WT and PNPLA2-KO schizonts that had been treated with DMSO (solvent control), 200 nM or 1 µM of proguanil were stained with the mitochondrial potentiometric dye rhodamine123 (Rho123, green) and parasites with a strong, weak or absent mitochondrial rhodamine123 signal were quantified by fluorescence microscopy. Shown are means +/- SD of four independent experiments, in which a total of 352 to 414 schizonts were analyzed per cell line and condition. For statistical evaluation a one-way ANOVA followed by a Holm-Sidak multiple comparison test was performed (*p

    Article Snippet: Phusion High-Fidelity DNA polymerase (New England BioLabs) was used for all plasmid constructions and all plasmid sequences were confirmed by Sanger sequencing.

    Techniques: Flow Cytometry, Positive Control, Staining, Fluorescence, Microscopy

    Sulfadiazine foliar spray selection of T 1 pCS4-GFP transgenic plants grown at a high density in soil . Sulfadiazine solution (500 mg/L with 0.03% L-77 silwet) was applied every third day for a total of three applications. A) Plant growth 21 days after the initial sulfadiazine application. B) Schematic representation of the CS4-GFP T-DNA, where the dashed line represents the region PCR amplified with sul I primers. C) PCR amplification results confirming the presence of sul I in 17 randomly chosen sulfadiazine resistant plants. The dash (-) indicates a water negative control, and the plus (+) indicates a plasmid positive control. A size standard labeled in kilobase pairs (kb) is shown.

    Journal: BMC Research Notes

    Article Title: Novel sulI binary vectors enable an inexpensive foliar selection method in Arabidopsis

    doi: 10.1186/1756-0500-4-44

    Figure Lengend Snippet: Sulfadiazine foliar spray selection of T 1 pCS4-GFP transgenic plants grown at a high density in soil . Sulfadiazine solution (500 mg/L with 0.03% L-77 silwet) was applied every third day for a total of three applications. A) Plant growth 21 days after the initial sulfadiazine application. B) Schematic representation of the CS4-GFP T-DNA, where the dashed line represents the region PCR amplified with sul I primers. C) PCR amplification results confirming the presence of sul I in 17 randomly chosen sulfadiazine resistant plants. The dash (-) indicates a water negative control, and the plus (+) indicates a plasmid positive control. A size standard labeled in kilobase pairs (kb) is shown.

    Article Snippet: Cc1 promoter isolation and pSUNG-OsCc1 construction A 1.8-kb fragment of the rice Cytochrome c gene (OsCc1 ) promoter was PCR-amplified (Phusion High-Fidelity DNA Polymerase, New England Biolabs) from Oryza sativa japonica cv.

    Techniques: Selection, Transgenic Assay, Polymerase Chain Reaction, Amplification, Negative Control, Plasmid Preparation, Positive Control, Labeling