polymerase chain reaction pcr mix  (Millipore)


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    Name:
    PCR Master
    Description:
    Routine assays have medium size amplicons and 50 GC content Taq DNA Polymerase has no proofreading or hot start features It is optimally active at 75C and pH 9 The 5 3 DNA polymerase lacks 3 5 exonuclease activity The thermostable enzyme is a single polypeptide chain with a molecular weight of approximately 95 kD Lack of restriction endonuclease The enzyme originally isolated from T aquaticus BM lacks Taq I restriction endonuclease activity
    Catalog Number:
    11636103001
    Price:
    None
    Applications:
    The PCR Master can be used instead of the single reagent Taq DNA Polmerase, or instead of the PCR Core Kit for nearly all simple, routine polymerase chain reaction applications. The only limitation is that the sample volume must not exceed half the total reaction volume. The PCR master mix is used for:. Routine PCR. RT-PCR. Other primer-extension reactions, such as sequencing and labeling
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    Structured Review

    Millipore polymerase chain reaction pcr mix
    PCR Master
    Routine assays have medium size amplicons and 50 GC content Taq DNA Polymerase has no proofreading or hot start features It is optimally active at 75C and pH 9 The 5 3 DNA polymerase lacks 3 5 exonuclease activity The thermostable enzyme is a single polypeptide chain with a molecular weight of approximately 95 kD Lack of restriction endonuclease The enzyme originally isolated from T aquaticus BM lacks Taq I restriction endonuclease activity
    https://www.bioz.com/result/polymerase chain reaction pcr mix/product/Millipore
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    polymerase chain reaction pcr mix - by Bioz Stars, 2020-08
    92/100 stars

    Images

    1) Product Images from "Immortalized mouse dental papilla mesenchymal cells preserve odontoblastic phenotype and respond to bone morphogenetic protein 2"

    Article Title: Immortalized mouse dental papilla mesenchymal cells preserve odontoblastic phenotype and respond to bone morphogenetic protein 2

    Journal: In Vitro Cellular & Developmental Biology. Animal

    doi: 10.1007/s11626-013-9641-1

    Expression of tooth-related genes in the primary and immortalized dental papilla mesenchymal cells. ( A ) Total RNAs from the primary and immortalized cells were extracted and reversely transcribed. The cDNAs were amplified by PCR using specific primers shown in Table 1 . The PCR products were run on 1% agarose gels and stained with ethidium bromide. M DNA marker (Low DNA mass ladder, Invitrogen), Con negative control. ( B ) Immunohistochemistry staining with antibodies against Col1a1, Dlx3, Dmp1, Dsp, Oc, Opn, Osx, and Runx2 in the primary and immortalized murine dental papilla mesenchymal cells. Scale bar = 10 μM. ( C ). Expression of Dsp and Dmp1 proteins in the primary and immortalized mouse dental papilla mesenchymal cells was analyzed by Western blot assay using anti-Dsp and anti-Dmp1 antibodies. Two major fragments of Dmp1 were observed as 57- and 37-kDa, respectively, in a SDS-PAGE gele using anti-Dmp1 antibody. Multiple fragments of Dsp polypeptides were detected in MD10-F2 cells using anti-Dsp antibodiy. Major bands are approximately 120, 65, 60, and 40 kDa as indicated by arrows . β-actin was used as an internal control. Pri and Imm represent the primary and immoratalized dental papilla mesenchymal cells.
    Figure Legend Snippet: Expression of tooth-related genes in the primary and immortalized dental papilla mesenchymal cells. ( A ) Total RNAs from the primary and immortalized cells were extracted and reversely transcribed. The cDNAs were amplified by PCR using specific primers shown in Table 1 . The PCR products were run on 1% agarose gels and stained with ethidium bromide. M DNA marker (Low DNA mass ladder, Invitrogen), Con negative control. ( B ) Immunohistochemistry staining with antibodies against Col1a1, Dlx3, Dmp1, Dsp, Oc, Opn, Osx, and Runx2 in the primary and immortalized murine dental papilla mesenchymal cells. Scale bar = 10 μM. ( C ). Expression of Dsp and Dmp1 proteins in the primary and immortalized mouse dental papilla mesenchymal cells was analyzed by Western blot assay using anti-Dsp and anti-Dmp1 antibodies. Two major fragments of Dmp1 were observed as 57- and 37-kDa, respectively, in a SDS-PAGE gele using anti-Dmp1 antibody. Multiple fragments of Dsp polypeptides were detected in MD10-F2 cells using anti-Dsp antibodiy. Major bands are approximately 120, 65, 60, and 40 kDa as indicated by arrows . β-actin was used as an internal control. Pri and Imm represent the primary and immoratalized dental papilla mesenchymal cells.

    Techniques Used: Expressing, Amplification, Polymerase Chain Reaction, Staining, Marker, Negative Control, Immunohistochemistry, Western Blot, SDS Page

    Identification of SV40 transformation. ( A ) Genomic DNA in the primary and immortalized dental papilla mesenchymal cells was isolated and amplified by the SV40 specific primer. PCR products were run on an agarose gel and stained with ethidium bromide. Lane 1 , DNA marker (Low DNA mass ladder, Invitrogen); lane 2 , control; lane 3 , primary dental papilla mesenchymal cells; lane 4 , immortalized cells. ( B , C ) Immunohistochemical staining with antibody against simian virus 40 large T-antigen (SV40 T-Ag) in iMDP-3 cells and immunolabeling of SV40 protein was mostly present in the nucleus ( green color ; B ) whereas immunostaining was not seen in the primary cells ( C ). The cells were stained with DAPI for the nucleus. Con control, Pri primary, Imm immortalized.
    Figure Legend Snippet: Identification of SV40 transformation. ( A ) Genomic DNA in the primary and immortalized dental papilla mesenchymal cells was isolated and amplified by the SV40 specific primer. PCR products were run on an agarose gel and stained with ethidium bromide. Lane 1 , DNA marker (Low DNA mass ladder, Invitrogen); lane 2 , control; lane 3 , primary dental papilla mesenchymal cells; lane 4 , immortalized cells. ( B , C ) Immunohistochemical staining with antibody against simian virus 40 large T-antigen (SV40 T-Ag) in iMDP-3 cells and immunolabeling of SV40 protein was mostly present in the nucleus ( green color ; B ) whereas immunostaining was not seen in the primary cells ( C ). The cells were stained with DAPI for the nucleus. Con control, Pri primary, Imm immortalized.

    Techniques Used: Transformation Assay, Isolation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Marker, Immunohistochemistry, Immunolabeling, Immunostaining

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells
    Article Snippet: .. The direct qPCR master mix includes a typical, real-time PCR master mix and other components, such as bovine serum albumin (BSA; Sigma, St. Louis, MO, USA), to adsorb PCR inhibitors from the test samples, a non-ionic detergent to modify the structures and dissolve the proteins of bacterial cell walls, and a calcium chelating agent . .. We had initially determined that this direct qPCR master mix (including the intermediate direct qPCR master mix) is more specialized than normal PCR master mix in adsorbing many of the PCR inhibitors present in samples .

    Magnetic Beads:

    Article Title: Detection of Mycobacterium bovis in Bovine Clinical Specimens Using Real-Time Fluorescence and Fluorescence Resonance Energy Transfer Probe Rapid-Cycle PCR
    Article Snippet: .. HBTC-PCR included either 2.5 μl of sequence-captured magnetic beads or a 5-μl inoculum of either genomic DNA or heat-inactivated M. bovi s. A PCR master mix (50-μl final volume) contained 2.5 U of Taq polymerase (Sigma), a 500 nM concentration of each of the IS 6110 primers, 250 μM deoxynucleoside triphosphates, and 1.75 mM MgCl2 , with a mineral oil overlay (Sigma). .. Conditions for cycling were 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 68°C for 2 min, and 74°C for 1 min. Amplification mixtures were incubated for a further 7 min at 74°C, with a final 4°C soak.

    Concentration Assay:

    Article Title: Detection of Mycobacterium bovis in Bovine Clinical Specimens Using Real-Time Fluorescence and Fluorescence Resonance Energy Transfer Probe Rapid-Cycle PCR
    Article Snippet: .. HBTC-PCR included either 2.5 μl of sequence-captured magnetic beads or a 5-μl inoculum of either genomic DNA or heat-inactivated M. bovi s. A PCR master mix (50-μl final volume) contained 2.5 U of Taq polymerase (Sigma), a 500 nM concentration of each of the IS 6110 primers, 250 μM deoxynucleoside triphosphates, and 1.75 mM MgCl2 , with a mineral oil overlay (Sigma). .. Conditions for cycling were 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 68°C for 2 min, and 74°C for 1 min. Amplification mixtures were incubated for a further 7 min at 74°C, with a final 4°C soak.

    SYBR Green Assay:

    Article Title: Over-expression of Interleukin-6 enhances cell survival and transformed cell growth in human malignant cholangiocytes
    Article Snippet: .. Each 20-μl reaction mixture consisted of 2 μl of cDNA (50 ng/μl), 10 μl of 2× Universal SYBR Green PCR Master Mix (Sigma, St.Louis, MO), and 2 μl of 20 nM forward and reverse primers. .. Optimization was performed for each gene-specific primer prior to the experiment to confirm that primer concentrations and reaction conditions did not produce artefactual amplification signals in control tubes that did not contain any template.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Evaluation of an Alternative Recombinant Thermostable Thermus thermophilus (rTth)-Based Real-Time Reverse Transcription-PCR Kit for Detection of Rotavirus A
    Article Snippet: .. Here, we report evaluation of a one-step RT-PCR master mix kit (EMD Millipore Corporation, Billerica, MA, USA) for detection of the RVA NSP3 gene. .. RNA extraction with an MS2 bacteriophage RNA (ZeptoMetrix, Buffalo, NY, USA) internal process control (IPC) ( , ) was conducted as described previously.

    Polymerase Chain Reaction:

    Article Title: A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells
    Article Snippet: .. The direct qPCR master mix includes a typical, real-time PCR master mix and other components, such as bovine serum albumin (BSA; Sigma, St. Louis, MO, USA), to adsorb PCR inhibitors from the test samples, a non-ionic detergent to modify the structures and dissolve the proteins of bacterial cell walls, and a calcium chelating agent . .. We had initially determined that this direct qPCR master mix (including the intermediate direct qPCR master mix) is more specialized than normal PCR master mix in adsorbing many of the PCR inhibitors present in samples .

    Article Title: Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition
    Article Snippet: .. Primers were purchased from GCC biotech (India) while PCR master mix and hot start DNA polymerase was procured from Sigma, USA. .. Bhabhatron-II, a teletherapy machine from Panacea, Medical Technologies Pvt Ltd (Bangalore, India) with a dose rate of 1.24 Gy/min. was used as a source for γ ray (Co-60) irradiation.

    Article Title: Detection of Mycobacterium bovis in Bovine Clinical Specimens Using Real-Time Fluorescence and Fluorescence Resonance Energy Transfer Probe Rapid-Cycle PCR
    Article Snippet: .. HBTC-PCR included either 2.5 μl of sequence-captured magnetic beads or a 5-μl inoculum of either genomic DNA or heat-inactivated M. bovi s. A PCR master mix (50-μl final volume) contained 2.5 U of Taq polymerase (Sigma), a 500 nM concentration of each of the IS 6110 primers, 250 μM deoxynucleoside triphosphates, and 1.75 mM MgCl2 , with a mineral oil overlay (Sigma). .. Conditions for cycling were 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 68°C for 2 min, and 74°C for 1 min. Amplification mixtures were incubated for a further 7 min at 74°C, with a final 4°C soak.

    Article Title: Over-expression of Interleukin-6 enhances cell survival and transformed cell growth in human malignant cholangiocytes
    Article Snippet: .. Each 20-μl reaction mixture consisted of 2 μl of cDNA (50 ng/μl), 10 μl of 2× Universal SYBR Green PCR Master Mix (Sigma, St.Louis, MO), and 2 μl of 20 nM forward and reverse primers. .. Optimization was performed for each gene-specific primer prior to the experiment to confirm that primer concentrations and reaction conditions did not produce artefactual amplification signals in control tubes that did not contain any template.

    Article Title: The RhoGAP protein Deleted in Liver Cancer 3 (DLC3) is essential for adherens junctions integrity
    Article Snippet: .. The cDNA was then used as a template for PCR analysis with REDTaq PCR Master Mix (Sigma). ..

    Sequencing:

    Article Title: Detection of Mycobacterium bovis in Bovine Clinical Specimens Using Real-Time Fluorescence and Fluorescence Resonance Energy Transfer Probe Rapid-Cycle PCR
    Article Snippet: .. HBTC-PCR included either 2.5 μl of sequence-captured magnetic beads or a 5-μl inoculum of either genomic DNA or heat-inactivated M. bovi s. A PCR master mix (50-μl final volume) contained 2.5 U of Taq polymerase (Sigma), a 500 nM concentration of each of the IS 6110 primers, 250 μM deoxynucleoside triphosphates, and 1.75 mM MgCl2 , with a mineral oil overlay (Sigma). .. Conditions for cycling were 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 68°C for 2 min, and 74°C for 1 min. Amplification mixtures were incubated for a further 7 min at 74°C, with a final 4°C soak.

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  • 92
    Millipore drg sections
    qPCR data of <t>Cx43</t> mRNA expression in a peripheral nerve, where Cx43 is differentially expressed at three different time points and b <t>DRG,</t> where Cx43 is downregulated 6 days after CCI. c DRG: qPCR data of BDNF mRNA expression shows an upregulation 24 h and 6 days post CCI. Mean ± SD, *p
    Drg Sections, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/drg sections/product/Millipore
    Average 92 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
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    92/100 stars
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    88
    Millipore qrt pcr reaction mix
    Comparison of droplet <t>qRT-PCR</t> amplification curves generated using a standard qPCR machine and epifluorescence imaging Drops containing (A) 10 1 M gene RNA cpd were thermocycled using qPCR. The solid green line represents real-time continuous fluorescence measurements of drops at each cycle as determined by qPCR, whereas the blue dashed line represents fluorescence measurements of sampled individual drops at cycle numbers 1, 20, 22, 24, 26, 28, 30, and 40 as determined by epifluorescence microscopy. Shaded error bars represent one standard deviation. (B) Histograms of individual drop fluorescence values from the epifluorescence images for 10 1 M gene RNA cpd show an increase in the fluorescence intensity from cycle 20 to 30. Cycle 1 and 40 represent pre- and post-thermocycling. N represents the number of drops analyzed from multiple epifluorescence images for each cycle. The arrow in cycle 40 represents unamplified drops present in our low RNA loading condition. (C) 10 4 M gene RNA cpd were thermocycled using qPCR. The solid orange line represents continuous fluorescence measurements of drops at each cycle as determined by qPCR, whereas the red dashed line represents fluorescence measurements of individual drops at cycle numbers 1, 10, 15, and 40 as determined by epifluorescence microscopy. Shaded error bars represent one standard deviation. (D) Histograms of individual drop fluorescence values from the epifluorescence images for 10 4 M gene RNA cpd show an increase in the fluorescence intensity from cycle 15 to 40. Cycle 1 and 40 represent pre- and post-thermocycling. N represents the number of drops analyzed from multiple epifluorescence images for each cycle. (E) Changes in fluorescence as cycle numbers increase are observed from representative epifluorescence images of 10 1 or 10 4 M gene RNA cpd at cycles 22 and 28 and cycles 10 and 15, respectively.
    Qrt Pcr Reaction Mix, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore ctrnp complexes
    Generation of a floxed Pitx1 allele using Easi -CRISPR. a – d The Easi- CRISPR strategy. a The two parts of the CRISPR guideRNA (crRNA + tracrRNA) and Cas9 protein. Combining them generates a <t>ctRNP</t> complex. The term ctRNP used here was formerly known as a cloning-free CRISPR/Cas9 system [ 28 ]. b A long <t>ssDNA</t> donor derived from a floxed exon cassette (or knock-in cassette as in Fig. 3 ) is mixed with ctRNP(s) to obtain the final Easi- CRISPR reagent cocktail for zygote injection. c Injection of a floxed ssDNA donor with right and left ctRNPs into zygotes results in replacement of the target exon with the floxed exon. For targeted insertions (as in Fig. 3 ) only a single ctRNP is required. d Following microinjection of the Easi -CRISPR reagent cocktail, genotyping and sequencing are used to identify founders with correctly modified genomes. e , f The Pitx1 wild-type allele, the Pitx1 ssDNA donor designed to flox exon 2 and the final targeted allele. The lengths of ssDNA, homology arms, and the distance between the two LoxP sites are shown. f Three genotyping PCRs and the primer combinations for these are indicated (5′ LoxP PCR, 5′ F + 5′ R primers; 3′ LoxP PCR, 3′ F + 3′ R primers; and full-length PCR, 5′ F + 3′ R primers). g Genotyping gel images from the ears of G0 offspring. The expected sizes of PCR amplicons (wild type ( wt ) or floxed) are indicated to the left of the gels. h Genotype interpretations are summarized below the gel image ( M monoallelic, P partial insertion, N no insertion). Animals 3, 5, 7, and 8 had both the 5′ and 3′ LoxP sites in cis , while animals 2, 4, 9, and 10 contained only one LoxP site, due to partial insertion of the ssDNA cassette
    Ctrnp Complexes, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore pcr mixtures
    <t>CDCE</t> profiles obtained from gills of the shipworm L. pedicellatus after <t>PCR</t> amplification with primers 948f and 1063r-GC. (A) Combined gills from four specimens used for the MSL but not included among the 13 individual specimens examined. (B to H) CDCE profiles from gills of seven representative specimens of L. pedicellatus , each showing a different combination of identified peaks. The identities of peaks P1 to P5 were confirmed by comigration with PCR products from sequenced clones spiked into samples after PCR and prior to CDCE. U1 in panel E represents an unidentified peak observed in one specimen only.
    Pcr Mixtures, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    qPCR data of Cx43 mRNA expression in a peripheral nerve, where Cx43 is differentially expressed at three different time points and b DRG, where Cx43 is downregulated 6 days after CCI. c DRG: qPCR data of BDNF mRNA expression shows an upregulation 24 h and 6 days post CCI. Mean ± SD, *p

    Journal: Molecular Pain

    Article Title: Expression changes of microRNA-1 and its targets Connexin 43 and brain-derived neurotrophic factor in the peripheral nervous system of chronic neuropathic rats

    doi: 10.1186/s12990-015-0045-y

    Figure Lengend Snippet: qPCR data of Cx43 mRNA expression in a peripheral nerve, where Cx43 is differentially expressed at three different time points and b DRG, where Cx43 is downregulated 6 days after CCI. c DRG: qPCR data of BDNF mRNA expression shows an upregulation 24 h and 6 days post CCI. Mean ± SD, *p

    Article Snippet: Cross sections of sciatic nerves were incubated with Cx43 (Cx43, ab11370, abcam, Cambridge, UK, 1:1,000), DRG sections were incubated with Cx43 and NeuN antibodies (Anti-NeuN, clone A60, Cat. # MAB377, Millipore, Temecula, USA) at 4°C overnight.

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Immunohistochemical staining of Cx43 in DRG at lower ( a , b , scale bar = 100 µm) and higher ( c , d , scale bar = 20 µm) magnification. Cx43 is located at the plasma membrane of large and small diameter neurons in Sham ( a , c ) and CCI (12 days) rats ( b , d ). NeuN neuronal cell marker, Hoechst nuclear counterstain.

    Journal: Molecular Pain

    Article Title: Expression changes of microRNA-1 and its targets Connexin 43 and brain-derived neurotrophic factor in the peripheral nervous system of chronic neuropathic rats

    doi: 10.1186/s12990-015-0045-y

    Figure Lengend Snippet: Immunohistochemical staining of Cx43 in DRG at lower ( a , b , scale bar = 100 µm) and higher ( c , d , scale bar = 20 µm) magnification. Cx43 is located at the plasma membrane of large and small diameter neurons in Sham ( a , c ) and CCI (12 days) rats ( b , d ). NeuN neuronal cell marker, Hoechst nuclear counterstain.

    Article Snippet: Cross sections of sciatic nerves were incubated with Cx43 (Cx43, ab11370, abcam, Cambridge, UK, 1:1,000), DRG sections were incubated with Cx43 and NeuN antibodies (Anti-NeuN, clone A60, Cat. # MAB377, Millipore, Temecula, USA) at 4°C overnight.

    Techniques: Immunohistochemistry, Staining, Marker

    a Protein expression of Cx43 in sciatic nerve; b expression of BDNF protein in sciatic nerve; c protein expression of Cx43 in DRG; d expression of BDNF protein in DRG. All experiments compare rats 12 days after CCI to Sham. Data show a marked upregulation of Cx43 and BDNF protein levels in nerve and DRG following CCI. Mean ± SD, *p

    Journal: Molecular Pain

    Article Title: Expression changes of microRNA-1 and its targets Connexin 43 and brain-derived neurotrophic factor in the peripheral nervous system of chronic neuropathic rats

    doi: 10.1186/s12990-015-0045-y

    Figure Lengend Snippet: a Protein expression of Cx43 in sciatic nerve; b expression of BDNF protein in sciatic nerve; c protein expression of Cx43 in DRG; d expression of BDNF protein in DRG. All experiments compare rats 12 days after CCI to Sham. Data show a marked upregulation of Cx43 and BDNF protein levels in nerve and DRG following CCI. Mean ± SD, *p

    Article Snippet: Cross sections of sciatic nerves were incubated with Cx43 (Cx43, ab11370, abcam, Cambridge, UK, 1:1,000), DRG sections were incubated with Cx43 and NeuN antibodies (Anti-NeuN, clone A60, Cat. # MAB377, Millipore, Temecula, USA) at 4°C overnight.

    Techniques: Expressing

    Comparison of droplet qRT-PCR amplification curves generated using a standard qPCR machine and epifluorescence imaging Drops containing (A) 10 1 M gene RNA cpd were thermocycled using qPCR. The solid green line represents real-time continuous fluorescence measurements of drops at each cycle as determined by qPCR, whereas the blue dashed line represents fluorescence measurements of sampled individual drops at cycle numbers 1, 20, 22, 24, 26, 28, 30, and 40 as determined by epifluorescence microscopy. Shaded error bars represent one standard deviation. (B) Histograms of individual drop fluorescence values from the epifluorescence images for 10 1 M gene RNA cpd show an increase in the fluorescence intensity from cycle 20 to 30. Cycle 1 and 40 represent pre- and post-thermocycling. N represents the number of drops analyzed from multiple epifluorescence images for each cycle. The arrow in cycle 40 represents unamplified drops present in our low RNA loading condition. (C) 10 4 M gene RNA cpd were thermocycled using qPCR. The solid orange line represents continuous fluorescence measurements of drops at each cycle as determined by qPCR, whereas the red dashed line represents fluorescence measurements of individual drops at cycle numbers 1, 10, 15, and 40 as determined by epifluorescence microscopy. Shaded error bars represent one standard deviation. (D) Histograms of individual drop fluorescence values from the epifluorescence images for 10 4 M gene RNA cpd show an increase in the fluorescence intensity from cycle 15 to 40. Cycle 1 and 40 represent pre- and post-thermocycling. N represents the number of drops analyzed from multiple epifluorescence images for each cycle. (E) Changes in fluorescence as cycle numbers increase are observed from representative epifluorescence images of 10 1 or 10 4 M gene RNA cpd at cycles 22 and 28 and cycles 10 and 15, respectively.

    Journal: bioRxiv

    Article Title: Screening of additives for droplet qRT-PCR thermocycling enables single influenza A virus genome quantification

    doi: 10.1101/2020.04.28.065342

    Figure Lengend Snippet: Comparison of droplet qRT-PCR amplification curves generated using a standard qPCR machine and epifluorescence imaging Drops containing (A) 10 1 M gene RNA cpd were thermocycled using qPCR. The solid green line represents real-time continuous fluorescence measurements of drops at each cycle as determined by qPCR, whereas the blue dashed line represents fluorescence measurements of sampled individual drops at cycle numbers 1, 20, 22, 24, 26, 28, 30, and 40 as determined by epifluorescence microscopy. Shaded error bars represent one standard deviation. (B) Histograms of individual drop fluorescence values from the epifluorescence images for 10 1 M gene RNA cpd show an increase in the fluorescence intensity from cycle 20 to 30. Cycle 1 and 40 represent pre- and post-thermocycling. N represents the number of drops analyzed from multiple epifluorescence images for each cycle. The arrow in cycle 40 represents unamplified drops present in our low RNA loading condition. (C) 10 4 M gene RNA cpd were thermocycled using qPCR. The solid orange line represents continuous fluorescence measurements of drops at each cycle as determined by qPCR, whereas the red dashed line represents fluorescence measurements of individual drops at cycle numbers 1, 10, 15, and 40 as determined by epifluorescence microscopy. Shaded error bars represent one standard deviation. (D) Histograms of individual drop fluorescence values from the epifluorescence images for 10 4 M gene RNA cpd show an increase in the fluorescence intensity from cycle 15 to 40. Cycle 1 and 40 represent pre- and post-thermocycling. N represents the number of drops analyzed from multiple epifluorescence images for each cycle. (E) Changes in fluorescence as cycle numbers increase are observed from representative epifluorescence images of 10 1 or 10 4 M gene RNA cpd at cycles 22 and 28 and cycles 10 and 15, respectively.

    Article Snippet: Tested additives were added to the qRT-PCR reaction mix at the following concentrations: 1% w/v Tween-20 (Calbiochem 655204-100mL), 0.8 µg/µL BSA (Fisher BP675-1), 2.5% w/v PEG-6K (Acros Organics 192280010), and 1 M betaine (Sigma B0300-1VL).

    Techniques: Quantitative RT-PCR, Amplification, Generated, Real-time Polymerase Chain Reaction, Imaging, Fluorescence, Epifluorescence Microscopy, Standard Deviation

    IAV amplified using droplet qRT-PCR method (A) Supernatant from infected cells was diluted and combined in drops with our optimized PCR mixture. The fraction of amplified drops is measured as the fraction of the number of fluorescent drops ( N + ) and the total number of drops ( N total ). A ddPCR analysis of end point epifluorescence imaging shows the dilution series of viral RNA in drops (solid blue line) coincides with the Poisson fit (dashed green line). Detection of viral RNA was achieved over four orders of magnitude before reaching the level of background amplification as determined using supernatant from mock infected cells (dashed red line). (B-E) Representative epifluorescence images of thermocycled drops containing various dilutions of viral supernatant from infected cells, including a mock sample with no virus.

    Journal: bioRxiv

    Article Title: Screening of additives for droplet qRT-PCR thermocycling enables single influenza A virus genome quantification

    doi: 10.1101/2020.04.28.065342

    Figure Lengend Snippet: IAV amplified using droplet qRT-PCR method (A) Supernatant from infected cells was diluted and combined in drops with our optimized PCR mixture. The fraction of amplified drops is measured as the fraction of the number of fluorescent drops ( N + ) and the total number of drops ( N total ). A ddPCR analysis of end point epifluorescence imaging shows the dilution series of viral RNA in drops (solid blue line) coincides with the Poisson fit (dashed green line). Detection of viral RNA was achieved over four orders of magnitude before reaching the level of background amplification as determined using supernatant from mock infected cells (dashed red line). (B-E) Representative epifluorescence images of thermocycled drops containing various dilutions of viral supernatant from infected cells, including a mock sample with no virus.

    Article Snippet: Tested additives were added to the qRT-PCR reaction mix at the following concentrations: 1% w/v Tween-20 (Calbiochem 655204-100mL), 0.8 µg/µL BSA (Fisher BP675-1), 2.5% w/v PEG-6K (Acros Organics 192280010), and 1 M betaine (Sigma B0300-1VL).

    Techniques: Amplification, Quantitative RT-PCR, Infection, Polymerase Chain Reaction, Imaging

    qRT-PCR dilution series of in vitro transcribed IAV M gene in drops (A) Amplification curves of six 10-fold dilutions of M gene RNA amplified in drops ranging from 10 −1 to 10 4 copies of RNA per drop (cpd). (B) C t standard curves for the bulk and drop amplification curves. The calculated qRT-PCR reaction efficiency was 90.3% for the drop dilution series and 98.9% for the bulk dilutions series, which falls within the desired range (90% to 110%). Representative epifluorescence images of the FAM and corresponding ROX channels of drops containing (C) 10 4 cpd and (D) 10 −1 cpd after 40 thermocycles. (E) Droplet digital PCR (ddPCR) analysis displaying the percentage of amplified drops as a function of RNA cpd. The total percentage of amplifying bright drops (red circles) increases as a function of RNA concentration and closely follows the Poisson estimate (blue dotted line).

    Journal: bioRxiv

    Article Title: Screening of additives for droplet qRT-PCR thermocycling enables single influenza A virus genome quantification

    doi: 10.1101/2020.04.28.065342

    Figure Lengend Snippet: qRT-PCR dilution series of in vitro transcribed IAV M gene in drops (A) Amplification curves of six 10-fold dilutions of M gene RNA amplified in drops ranging from 10 −1 to 10 4 copies of RNA per drop (cpd). (B) C t standard curves for the bulk and drop amplification curves. The calculated qRT-PCR reaction efficiency was 90.3% for the drop dilution series and 98.9% for the bulk dilutions series, which falls within the desired range (90% to 110%). Representative epifluorescence images of the FAM and corresponding ROX channels of drops containing (C) 10 4 cpd and (D) 10 −1 cpd after 40 thermocycles. (E) Droplet digital PCR (ddPCR) analysis displaying the percentage of amplified drops as a function of RNA cpd. The total percentage of amplifying bright drops (red circles) increases as a function of RNA concentration and closely follows the Poisson estimate (blue dotted line).

    Article Snippet: Tested additives were added to the qRT-PCR reaction mix at the following concentrations: 1% w/v Tween-20 (Calbiochem 655204-100mL), 0.8 µg/µL BSA (Fisher BP675-1), 2.5% w/v PEG-6K (Acros Organics 192280010), and 1 M betaine (Sigma B0300-1VL).

    Techniques: Quantitative RT-PCR, In Vitro, Amplification, Digital PCR, Concentration Assay

    Generation of a floxed Pitx1 allele using Easi -CRISPR. a – d The Easi- CRISPR strategy. a The two parts of the CRISPR guideRNA (crRNA + tracrRNA) and Cas9 protein. Combining them generates a ctRNP complex. The term ctRNP used here was formerly known as a cloning-free CRISPR/Cas9 system [ 28 ]. b A long ssDNA donor derived from a floxed exon cassette (or knock-in cassette as in Fig. 3 ) is mixed with ctRNP(s) to obtain the final Easi- CRISPR reagent cocktail for zygote injection. c Injection of a floxed ssDNA donor with right and left ctRNPs into zygotes results in replacement of the target exon with the floxed exon. For targeted insertions (as in Fig. 3 ) only a single ctRNP is required. d Following microinjection of the Easi -CRISPR reagent cocktail, genotyping and sequencing are used to identify founders with correctly modified genomes. e , f The Pitx1 wild-type allele, the Pitx1 ssDNA donor designed to flox exon 2 and the final targeted allele. The lengths of ssDNA, homology arms, and the distance between the two LoxP sites are shown. f Three genotyping PCRs and the primer combinations for these are indicated (5′ LoxP PCR, 5′ F + 5′ R primers; 3′ LoxP PCR, 3′ F + 3′ R primers; and full-length PCR, 5′ F + 3′ R primers). g Genotyping gel images from the ears of G0 offspring. The expected sizes of PCR amplicons (wild type ( wt ) or floxed) are indicated to the left of the gels. h Genotype interpretations are summarized below the gel image ( M monoallelic, P partial insertion, N no insertion). Animals 3, 5, 7, and 8 had both the 5′ and 3′ LoxP sites in cis , while animals 2, 4, 9, and 10 contained only one LoxP site, due to partial insertion of the ssDNA cassette

    Journal: Genome Biology

    Article Title: Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

    doi: 10.1186/s13059-017-1220-4

    Figure Lengend Snippet: Generation of a floxed Pitx1 allele using Easi -CRISPR. a – d The Easi- CRISPR strategy. a The two parts of the CRISPR guideRNA (crRNA + tracrRNA) and Cas9 protein. Combining them generates a ctRNP complex. The term ctRNP used here was formerly known as a cloning-free CRISPR/Cas9 system [ 28 ]. b A long ssDNA donor derived from a floxed exon cassette (or knock-in cassette as in Fig. 3 ) is mixed with ctRNP(s) to obtain the final Easi- CRISPR reagent cocktail for zygote injection. c Injection of a floxed ssDNA donor with right and left ctRNPs into zygotes results in replacement of the target exon with the floxed exon. For targeted insertions (as in Fig. 3 ) only a single ctRNP is required. d Following microinjection of the Easi -CRISPR reagent cocktail, genotyping and sequencing are used to identify founders with correctly modified genomes. e , f The Pitx1 wild-type allele, the Pitx1 ssDNA donor designed to flox exon 2 and the final targeted allele. The lengths of ssDNA, homology arms, and the distance between the two LoxP sites are shown. f Three genotyping PCRs and the primer combinations for these are indicated (5′ LoxP PCR, 5′ F + 5′ R primers; 3′ LoxP PCR, 3′ F + 3′ R primers; and full-length PCR, 5′ F + 3′ R primers). g Genotyping gel images from the ears of G0 offspring. The expected sizes of PCR amplicons (wild type ( wt ) or floxed) are indicated to the left of the gels. h Genotype interpretations are summarized below the gel image ( M monoallelic, P partial insertion, N no insertion). Animals 3, 5, 7, and 8 had both the 5′ and 3′ LoxP sites in cis , while animals 2, 4, 9, and 10 contained only one LoxP site, due to partial insertion of the ssDNA cassette

    Article Snippet: The ssDNA donors were mixed with ctRNP complexes at 5–10 ng/mix and the final injection mixes were passed through Millipore Centrifugal Filter units (UFC30VV25, EMD Millipore, Billerica, MA, USA) and spun at 21,000 g for 5 min at room temperature.

    Techniques: CRISPR, Clone Assay, Derivative Assay, Knock-In, Injection, Sequencing, Modification, Polymerase Chain Reaction

    CDCE profiles obtained from gills of the shipworm L. pedicellatus after PCR amplification with primers 948f and 1063r-GC. (A) Combined gills from four specimens used for the MSL but not included among the 13 individual specimens examined. (B to H) CDCE profiles from gills of seven representative specimens of L. pedicellatus , each showing a different combination of identified peaks. The identities of peaks P1 to P5 were confirmed by comigration with PCR products from sequenced clones spiked into samples after PCR and prior to CDCE. U1 in panel E represents an unidentified peak observed in one specimen only.

    Journal: Applied and Environmental Microbiology

    Article Title: Extensive Variation in Intracellular Symbiont Community Composition among Members of a Single Population of the Wood-Boring Bivalve Lyrodus pedicellatus (Bivalvia: Teredinidae)

    doi: 10.1128/AEM.72.1.412-417.2006

    Figure Lengend Snippet: CDCE profiles obtained from gills of the shipworm L. pedicellatus after PCR amplification with primers 948f and 1063r-GC. (A) Combined gills from four specimens used for the MSL but not included among the 13 individual specimens examined. (B to H) CDCE profiles from gills of seven representative specimens of L. pedicellatus , each showing a different combination of identified peaks. The identities of peaks P1 to P5 were confirmed by comigration with PCR products from sequenced clones spiked into samples after PCR and prior to CDCE. U1 in panel E represents an unidentified peak observed in one specimen only.

    Article Snippet: For CDCE analysis, PCR mixtures were diluted 50-fold into distilled and deionized water (Millipore, Billerica, MA) and electroinjected for 45 s at 2 μA into a fused silica capillary (75-μm inside diameter) filled with a replaceable 5% linear polyacrylamide gel matrix (Scientific Polymers, Ontario, NY).

    Techniques: Polymerase Chain Reaction, Amplification, Clone Assay