hot start dna polymerase  (Millipore)


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    Name:
    DNA Polymerase
    Description:
    Thermo Sequenase is a novel thermostable DNA polymerase that uses dideoxynucleotide triphosphates ddNTPs as readily as deoxynucleotide dNTP substrates Uniform and easy to read band patterns Thermostable enzyme Thermo Sequenase is a novel thermostable DNA polymerase that uses dideoxynucleotide triphosphates ddNTPs as readily as deoxynucleotide dNTP substrates This results in extremely uniform and easy to read sequence band patterns This property in addition to thermostability makes Thermo Sequenase an excellent choice for cycle sequencing where template quantity limitations preclude the use of T7 Sequenase DNA polymerase The properties of Thermo Sequenase also enable its use in many SNP genotyping applications where primer extension is employed
    Catalog Number:
    gee79000z
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    Structured Review

    Millipore hot start dna polymerase
    Radiation induced mitochondrial biogenesis augments metabolic viability: ( A ) Cell cycle histogram showing phase distribution (G1, S and G2/M) of cells at 24 and 48 hrs post irradiation in HeLa and MDA-MB-231 cells. ( B ) Mitochondrial genome encoded Leu tRNA gene was analyzed by semi quantitative <t>PCR</t> and normalized with nuclear pol gamma gene copy number. The mtDNA copy number is also presented as comparative fold change at respective time points (bar diagram). ( C ) Protein expression analysis of mitochondrial biogenesis and mitochondrial complex-II subunit SDH-A presented in HeLa and MDA-MB-231 cells. The values between the blots represent the fold increase at 8 and 24 hrs. post irradiation quantified by densitometry and normalized with respective β-Actin. The <t>DNA</t> ( B ) and protein blot ( C ). ( D ) Analysis of effect of chloramphenicol (40 μM; 30 mins prior to IR; continuous exposure) on mitochondrial content by MitoTracker Green FM at indicated time points using flow cytometer and graphs presented as fold change of mean fluorescence intensity (MFI) with respective control. Effect of Chloramphenicol on radiation induced growth inhibition was analyzed (at 5 Gy) by MTT assay ( E ) and cell number ( F ) in HeLa and MDA-MB-231 cells. Growth inhibition quantified and mentioned at 48 hours. Data are expressed as mean ± SD from triplicates. *p
    Thermo Sequenase is a novel thermostable DNA polymerase that uses dideoxynucleotide triphosphates ddNTPs as readily as deoxynucleotide dNTP substrates Uniform and easy to read band patterns Thermostable enzyme Thermo Sequenase is a novel thermostable DNA polymerase that uses dideoxynucleotide triphosphates ddNTPs as readily as deoxynucleotide dNTP substrates This results in extremely uniform and easy to read sequence band patterns This property in addition to thermostability makes Thermo Sequenase an excellent choice for cycle sequencing where template quantity limitations preclude the use of T7 Sequenase DNA polymerase The properties of Thermo Sequenase also enable its use in many SNP genotyping applications where primer extension is employed
    https://www.bioz.com/result/hot start dna polymerase/product/Millipore
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    hot start dna polymerase - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition"

    Article Title: Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19930-w

    Radiation induced mitochondrial biogenesis augments metabolic viability: ( A ) Cell cycle histogram showing phase distribution (G1, S and G2/M) of cells at 24 and 48 hrs post irradiation in HeLa and MDA-MB-231 cells. ( B ) Mitochondrial genome encoded Leu tRNA gene was analyzed by semi quantitative PCR and normalized with nuclear pol gamma gene copy number. The mtDNA copy number is also presented as comparative fold change at respective time points (bar diagram). ( C ) Protein expression analysis of mitochondrial biogenesis and mitochondrial complex-II subunit SDH-A presented in HeLa and MDA-MB-231 cells. The values between the blots represent the fold increase at 8 and 24 hrs. post irradiation quantified by densitometry and normalized with respective β-Actin. The DNA ( B ) and protein blot ( C ). ( D ) Analysis of effect of chloramphenicol (40 μM; 30 mins prior to IR; continuous exposure) on mitochondrial content by MitoTracker Green FM at indicated time points using flow cytometer and graphs presented as fold change of mean fluorescence intensity (MFI) with respective control. Effect of Chloramphenicol on radiation induced growth inhibition was analyzed (at 5 Gy) by MTT assay ( E ) and cell number ( F ) in HeLa and MDA-MB-231 cells. Growth inhibition quantified and mentioned at 48 hours. Data are expressed as mean ± SD from triplicates. *p
    Figure Legend Snippet: Radiation induced mitochondrial biogenesis augments metabolic viability: ( A ) Cell cycle histogram showing phase distribution (G1, S and G2/M) of cells at 24 and 48 hrs post irradiation in HeLa and MDA-MB-231 cells. ( B ) Mitochondrial genome encoded Leu tRNA gene was analyzed by semi quantitative PCR and normalized with nuclear pol gamma gene copy number. The mtDNA copy number is also presented as comparative fold change at respective time points (bar diagram). ( C ) Protein expression analysis of mitochondrial biogenesis and mitochondrial complex-II subunit SDH-A presented in HeLa and MDA-MB-231 cells. The values between the blots represent the fold increase at 8 and 24 hrs. post irradiation quantified by densitometry and normalized with respective β-Actin. The DNA ( B ) and protein blot ( C ). ( D ) Analysis of effect of chloramphenicol (40 μM; 30 mins prior to IR; continuous exposure) on mitochondrial content by MitoTracker Green FM at indicated time points using flow cytometer and graphs presented as fold change of mean fluorescence intensity (MFI) with respective control. Effect of Chloramphenicol on radiation induced growth inhibition was analyzed (at 5 Gy) by MTT assay ( E ) and cell number ( F ) in HeLa and MDA-MB-231 cells. Growth inhibition quantified and mentioned at 48 hours. Data are expressed as mean ± SD from triplicates. *p

    Techniques Used: Irradiation, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Fluorescence, Inhibition, MTT Assay

    2) Product Images from "Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition"

    Article Title: Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19930-w

    Radiation induced mitochondrial biogenesis augments metabolic viability: ( A ) Cell cycle histogram showing phase distribution (G1, S and G2/M) of cells at 24 and 48 hrs post irradiation in HeLa and MDA-MB-231 cells. ( B ) Mitochondrial genome encoded Leu tRNA gene was analyzed by semi quantitative PCR and normalized with nuclear pol gamma gene copy number. The mtDNA copy number is also presented as comparative fold change at respective time points (bar diagram). ( C ) Protein expression analysis of mitochondrial biogenesis and mitochondrial complex-II subunit SDH-A presented in HeLa and MDA-MB-231 cells. The values between the blots represent the fold increase at 8 and 24 hrs. post irradiation quantified by densitometry and normalized with respective β-Actin. The DNA ( B ) and protein blot ( C ) images were cropped from full-length blots (Supplementary Figs 2 and 3 ). ( D ) Analysis of effect of chloramphenicol (40 μM; 30 mins prior to IR; continuous exposure) on mitochondrial content by MitoTracker Green FM at indicated time points using flow cytometer and graphs presented as fold change of mean fluorescence intensity (MFI) with respective control. Effect of Chloramphenicol on radiation induced growth inhibition was analyzed (at 5 Gy) by MTT assay ( E ) and cell number ( F ) in HeLa and MDA-MB-231 cells. Growth inhibition quantified and mentioned at 48 hours. Data are expressed as mean ± SD from triplicates. *p
    Figure Legend Snippet: Radiation induced mitochondrial biogenesis augments metabolic viability: ( A ) Cell cycle histogram showing phase distribution (G1, S and G2/M) of cells at 24 and 48 hrs post irradiation in HeLa and MDA-MB-231 cells. ( B ) Mitochondrial genome encoded Leu tRNA gene was analyzed by semi quantitative PCR and normalized with nuclear pol gamma gene copy number. The mtDNA copy number is also presented as comparative fold change at respective time points (bar diagram). ( C ) Protein expression analysis of mitochondrial biogenesis and mitochondrial complex-II subunit SDH-A presented in HeLa and MDA-MB-231 cells. The values between the blots represent the fold increase at 8 and 24 hrs. post irradiation quantified by densitometry and normalized with respective β-Actin. The DNA ( B ) and protein blot ( C ) images were cropped from full-length blots (Supplementary Figs 2 and 3 ). ( D ) Analysis of effect of chloramphenicol (40 μM; 30 mins prior to IR; continuous exposure) on mitochondrial content by MitoTracker Green FM at indicated time points using flow cytometer and graphs presented as fold change of mean fluorescence intensity (MFI) with respective control. Effect of Chloramphenicol on radiation induced growth inhibition was analyzed (at 5 Gy) by MTT assay ( E ) and cell number ( F ) in HeLa and MDA-MB-231 cells. Growth inhibition quantified and mentioned at 48 hours. Data are expressed as mean ± SD from triplicates. *p

    Techniques Used: Irradiation, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Fluorescence, Inhibition, MTT Assay

    Related Articles

    Clone Assay:

    Article Title: Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements
    Article Snippet: These custom-made primers (Cortec DNA Service Laboratories Inc., Kingston, Canada) were used to generate polymerase chain reaction (PCR) fragments in reactions involving a DNA polymerase deficient in 3′–5′ exonuclease activity (KOD polymerase; Novagen, Gibbstown, NJ, USA) to minimise proofreading errors. .. Fragments generated, either by PCR or cloning in E.coli , were sequenced in both directions using an ABI Prism® 3100 Genetic Analyzer and BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).

    Amplification:

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae
    Article Snippet: Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore). .. Second, the assembled Cas9 gene cassette was PCR amplified and a second round of in vivo /10 promoter and downstream of the MX terminator.

    Article Title: Molecular Characterization of Natural Erwinia pyrifoliae Strains Deficient in Hypersensitive Response
    Article Snippet: .. To reconfirm the hrpL sequences, DNA from strains Ep2/97, Ep4/97, and Ep16/96 was also amplified with a proofreading DNA polymerase (Accu Taq ; Sigma). .. The same sequence as in Fig. was obtained for the three wild-type strains, and the change from C to T at position 277 for strain Ep2/97 was observed.

    Synthesized:

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae
    Article Snippet: The second Cas9* gene used for the tandem fusions was also synthesized after manual manipulation of each codon to an alternate codon (primarily within the Wobble position). .. Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore).

    Enzyme Activity Assay:

    Article Title: Towards the development of an enzyme replacement therapy for the metabolic disorder propionic acidemia
    Article Snippet: Restriction endonucleases (Hin dIII-HF, Not I-HF, Eco RI-HF and Bam HI-HF) and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA), while KOD DNA polymerase was purchased from Novagen-EMD4 Biosciences (Darmstadt, Germany). .. Mitochondrial isolation from mammalian cells and enzymatic activity assay were performed as described previously .

    Incubation:

    Article Title: Role of Androgen Receptor in Progression of LNCaP Prostate Cancer Cells from G1 to S Phase
    Article Snippet: .. Slides were then incubated for 1 hour at 22°C with antibodies against AR (AR-N20 or AR-441) and Cdc6 or DNA polymerase-α followed by both goat-anti-mouse-fluorescein isothiocyanate (FITC)- and goat-anti-rabbit-tetramethylrhodamine B isothiocyanate (TRITC)-labeled secondary antibodies (Sigma-Aldrich, St. Louis, MO). .. After four washes with PBS, slides were mounted with Aqua-Poly/Mount (Polysciences Inc., Warrington, PA).

    Article Title: Characterization of Family D DNA polymerase from Thermococcus sp. 9?N
    Article Snippet: To determine the effects of 9°N PCNA and 9°N RFC (hereafter referred to as PCNA and RFC) on polD activity, polD (22 nM) was incubated at 65 °C with PCNA (10 nM) and RFC (21 nM) in a reaction containing 100 µM dATP, dCTP, and TTP, 10 µM dGTP, 2 mM ATP, 0.02 µCi/µl [α-32 P]-dGTP, 0.5 nM primed M13mp18, 10 mM MgSO4 , 250 mM NaCl, and 40 mM Tris–HCl, pH 8.0. .. To test the effect of aphidicolin on polB and polD, the DNA polymerase assay described above was repeated in the presence or absence of aphidicolin (Sigma).

    Activity Assay:

    Article Title: Characterization of Family D DNA polymerase from Thermococcus sp. 9?N
    Article Snippet: Paragraph title: DNA polymerization activity assay ... To test the effect of aphidicolin on polB and polD, the DNA polymerase assay described above was repeated in the presence or absence of aphidicolin (Sigma).

    Article Title: Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements
    Article Snippet: .. These custom-made primers (Cortec DNA Service Laboratories Inc., Kingston, Canada) were used to generate polymerase chain reaction (PCR) fragments in reactions involving a DNA polymerase deficient in 3′–5′ exonuclease activity (KOD polymerase; Novagen, Gibbstown, NJ, USA) to minimise proofreading errors. .. Fragments generated, either by PCR or cloning in E.coli , were sequenced in both directions using an ABI Prism® 3100 Genetic Analyzer and BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).

    BIA-KA:

    Article Title: Identification of a Redox-Modulatory Interaction Between Uncoupling Protein 3 and Thioredoxin 2 in the Mitochondrial Intermembrane Space
    Article Snippet: The Pierce BCA Assay (Pierce Biotechnology, Rockford, IL) was used to quantify proteins. .. The proteins were transferred to a nitrocellulose membrane and probed with primary antibody according to the manufacturer's instructions; anti-V5, anti-UCP3, anti-aconitase 2, anti-β-actin (Abcam, Cambridge, MA), anti-cytochrome c (BD Biosciences, Franklin Lakes, NJ), anti-myc, anti-phospho-p38, anti-p38 (Cell Signaling), anti-COX4 (Clontech, Mountain View, CA), anti-DNA polymerase γ (Neo Markers, Fremont, CA), anti-T7 (Novagen, Gibbstown, NJ), and anti-thioredoxin 2 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were used.

    Modification:

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae
    Article Snippet: .. Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore). ..

    Ligation:

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae
    Article Snippet: Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore). .. Second, the assembled Cas9 gene cassette was PCR amplified and a second round of in vivo ligation was performed using a second vector (pGF-IVL974) to insert 992 bp of HIS3 5′ UTR, 993 bp 3′ UTR, and two (u2) sites ( ) upstream of the GAL1 /10 promoter and downstream of the MX terminator.

    Generated:

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae
    Article Snippet: .. Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore). ..

    Article Title: Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements
    Article Snippet: Sequence analysis and derivation of DNA probes Unless otherwise indicated, most of the DNA sequences used and generated in this work are identified by GenBank® accession numbers (see and , and legends to ). .. These custom-made primers (Cortec DNA Service Laboratories Inc., Kingston, Canada) were used to generate polymerase chain reaction (PCR) fragments in reactions involving a DNA polymerase deficient in 3′–5′ exonuclease activity (KOD polymerase; Novagen, Gibbstown, NJ, USA) to minimise proofreading errors.

    Article Title: Expression of Kir7.1 and a Novel Kir7.1 Splice Variant in Native Human Retinal Pigment Epithelium
    Article Snippet: .. For Kir7.1 and Kir7.1S primer sets 1 & 2 (see ), the PCR products were generated by adding DNA polymerase (AccuTaq LA DNA polymerase; Sigma, St. Louis, MO) and cycled 30 times (15 seconds at 94°C, 20 seconds at 52°C, 4 minutes at 68°C), followed by a 10 min-extension at 68°C. ..

    Confocal Laser Scanning Microscopy:

    Article Title: Role of Androgen Receptor in Progression of LNCaP Prostate Cancer Cells from G1 to S Phase
    Article Snippet: Slides were then incubated for 1 hour at 22°C with antibodies against AR (AR-N20 or AR-441) and Cdc6 or DNA polymerase-α followed by both goat-anti-mouse-fluorescein isothiocyanate (FITC)- and goat-anti-rabbit-tetramethylrhodamine B isothiocyanate (TRITC)-labeled secondary antibodies (Sigma-Aldrich, St. Louis, MO). .. Confocal laser scanning microscopy was performed with a Zeiss LSM 410 upright confocal microscope.

    Sequencing:

    Article Title: Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements
    Article Snippet: Paragraph title: Sequence analysis and derivation of DNA probes ... These custom-made primers (Cortec DNA Service Laboratories Inc., Kingston, Canada) were used to generate polymerase chain reaction (PCR) fragments in reactions involving a DNA polymerase deficient in 3′–5′ exonuclease activity (KOD polymerase; Novagen, Gibbstown, NJ, USA) to minimise proofreading errors.

    Article Title: Molecular Characterization of Natural Erwinia pyrifoliae Strains Deficient in Hypersensitive Response
    Article Snippet: The nucleotide sequence of hrp L from Ep16/96 corresponded with the nucleotide sequence from Ep4/97 with C at position 277. .. To reconfirm the hrpL sequences, DNA from strains Ep2/97, Ep4/97, and Ep16/96 was also amplified with a proofreading DNA polymerase (Accu Taq ; Sigma).

    MTT Assay:

    Article Title: Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition
    Article Snippet: Dulbecco’s Minimum Essential Medium (DMEM), Penicillin G, streptomycin, nystatin, dimethyl sulfoxide (DMSO), 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Ruthenium Red, and BAPTA-AM were purchased from Sigma Chemicals Co. (St Louis, USA), where as Chloroamphenicol was obtained from Amresco USA. .. Primers were purchased from GCC biotech (India) while PCR master mix and hot start DNA polymerase was procured from Sigma, USA.

    In Vivo:

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae
    Article Snippet: Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore). .. First, a CEN-based (pRS316) plasmid was generated by in vivo /10 terminator (238 bp) , and the MX-based kanamycin resistance cassette ( ).

    Radioactivity:

    Article Title: Synthesis and Enzymatic Incorporation of Modified Deoxyuridine Triphosphates
    Article Snippet: KOD-DASH DNA polymerase was purchased from EMD Millipore (Billerica, MA, USA). .. Radioactivity was visualized using a Typhoon 9200 variable mode imager from GE Healthcare (Velizy-Villacoublay, France).

    Mutagenesis:

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae
    Article Snippet: .. Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore). ..

    Isolation:

    Article Title: Towards the development of an enzyme replacement therapy for the metabolic disorder propionic acidemia
    Article Snippet: Restriction endonucleases (Hin dIII-HF, Not I-HF, Eco RI-HF and Bam HI-HF) and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA), while KOD DNA polymerase was purchased from Novagen-EMD4 Biosciences (Darmstadt, Germany). .. Mitochondrial isolation from mammalian cells and enzymatic activity assay were performed as described previously .

    Microscopy:

    Article Title: Role of Androgen Receptor in Progression of LNCaP Prostate Cancer Cells from G1 to S Phase
    Article Snippet: Slides were then incubated for 1 hour at 22°C with antibodies against AR (AR-N20 or AR-441) and Cdc6 or DNA polymerase-α followed by both goat-anti-mouse-fluorescein isothiocyanate (FITC)- and goat-anti-rabbit-tetramethylrhodamine B isothiocyanate (TRITC)-labeled secondary antibodies (Sigma-Aldrich, St. Louis, MO). .. Confocal laser scanning microscopy was performed with a Zeiss LSM 410 upright confocal microscope.

    Polymerase Chain Reaction:

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae
    Article Snippet: .. Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore). ..

    Article Title: Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements
    Article Snippet: .. These custom-made primers (Cortec DNA Service Laboratories Inc., Kingston, Canada) were used to generate polymerase chain reaction (PCR) fragments in reactions involving a DNA polymerase deficient in 3′–5′ exonuclease activity (KOD polymerase; Novagen, Gibbstown, NJ, USA) to minimise proofreading errors. .. Fragments generated, either by PCR or cloning in E.coli , were sequenced in both directions using an ABI Prism® 3100 Genetic Analyzer and BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).

    Article Title: Expression of Kir7.1 and a Novel Kir7.1 Splice Variant in Native Human Retinal Pigment Epithelium
    Article Snippet: .. For Kir7.1 and Kir7.1S primer sets 1 & 2 (see ), the PCR products were generated by adding DNA polymerase (AccuTaq LA DNA polymerase; Sigma, St. Louis, MO) and cycled 30 times (15 seconds at 94°C, 20 seconds at 52°C, 4 minutes at 68°C), followed by a 10 min-extension at 68°C. ..

    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.

    Labeling:

    Article Title: Molecular Characterization of Natural Erwinia pyrifoliae Strains Deficient in Hypersensitive Response
    Article Snippet: The insertions obtained from strains Ep2/97 and Ep4/97 were sequenced with labeled SP6 and T7 primers in both strands by using an ALF sequencer, and the nucleotide sequences were independently confirmed by commercial sequencing. .. To reconfirm the hrpL sequences, DNA from strains Ep2/97, Ep4/97, and Ep16/96 was also amplified with a proofreading DNA polymerase (Accu Taq ; Sigma).

    Confocal Microscopy:

    Article Title: Role of Androgen Receptor in Progression of LNCaP Prostate Cancer Cells from G1 to S Phase
    Article Snippet: Paragraph title: Immunofluorescent Staining and Confocal Microscopy ... Slides were then incubated for 1 hour at 22°C with antibodies against AR (AR-N20 or AR-441) and Cdc6 or DNA polymerase-α followed by both goat-anti-mouse-fluorescein isothiocyanate (FITC)- and goat-anti-rabbit-tetramethylrhodamine B isothiocyanate (TRITC)-labeled secondary antibodies (Sigma-Aldrich, St. Louis, MO).

    Plasmid Preparation:

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae
    Article Snippet: .. Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore). ..

    Article Title: Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements
    Article Snippet: The oligonucleotide primers (see list in ) were designed using Vector NTI™ version 9.0.0 software (Invitrogen, Burlington, Ontario, Canada) and GenBank® sequence data for λ bacteriophage, imm 434 and E.coli lacZ . .. These custom-made primers (Cortec DNA Service Laboratories Inc., Kingston, Canada) were used to generate polymerase chain reaction (PCR) fragments in reactions involving a DNA polymerase deficient in 3′–5′ exonuclease activity (KOD polymerase; Novagen, Gibbstown, NJ, USA) to minimise proofreading errors.

    Software:

    Article Title: Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements
    Article Snippet: The oligonucleotide primers (see list in ) were designed using Vector NTI™ version 9.0.0 software (Invitrogen, Burlington, Ontario, Canada) and GenBank® sequence data for λ bacteriophage, imm 434 and E.coli lacZ . .. These custom-made primers (Cortec DNA Service Laboratories Inc., Kingston, Canada) were used to generate polymerase chain reaction (PCR) fragments in reactions involving a DNA polymerase deficient in 3′–5′ exonuclease activity (KOD polymerase; Novagen, Gibbstown, NJ, USA) to minimise proofreading errors.

    Irradiation:

    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.

    Concentration Assay:

    Article Title: Characterization of Family D DNA polymerase from Thermococcus sp. 9?N
    Article Snippet: Aliquots were sampled and reactions stopped by the addition of EDTA (100 mM final concentration). .. To test the effect of aphidicolin on polB and polD, the DNA polymerase assay described above was repeated in the presence or absence of aphidicolin (Sigma).

    Staining:

    Article Title: Role of Androgen Receptor in Progression of LNCaP Prostate Cancer Cells from G1 to S Phase
    Article Snippet: Paragraph title: Immunofluorescent Staining and Confocal Microscopy ... Slides were then incubated for 1 hour at 22°C with antibodies against AR (AR-N20 or AR-441) and Cdc6 or DNA polymerase-α followed by both goat-anti-mouse-fluorescein isothiocyanate (FITC)- and goat-anti-rabbit-tetramethylrhodamine B isothiocyanate (TRITC)-labeled secondary antibodies (Sigma-Aldrich, St. Louis, MO).

    Article Title: Towards the development of an enzyme replacement therapy for the metabolic disorder propionic acidemia
    Article Snippet: GelRed® nucleic acid gel stain, MitoView 633 fluorescent mitochondrial dye, and Mix-n-Stain™ CF488A were purchased from Biotium (Hayward, CA). .. Restriction endonucleases (Hin dIII-HF, Not I-HF, Eco RI-HF and Bam HI-HF) and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA), while KOD DNA polymerase was purchased from Novagen-EMD4 Biosciences (Darmstadt, Germany).

    other:

    Article Title: Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿
    Article Snippet: The following commercial thermostable DNA polymerases were purchased: Pfu DNA polymerase (Stratagene), Vent DNA polymerase (New England Biolabs), KOD DNA polymerase (Novagen), and Taq DNA polymerase (Rexgene Biotech).

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    Millipore kod hot start dna polymerase
    Performance of four thermostable <t>DNA</t> polymerases for the synthesis of gene B using PCA-DTF. Lane 1: negative control reaction performed at the same conditions, without addition of primers; lane 2: <t>KOD</t> Hot Start DNA polymerase; lane 3: Q5 Hot Start High Fidelity DNA polymerase; lane 4: Pfu Turbo DNA polymerase and lane 5: Taq DNA polymerase. M2: NZYladder I
    Kod Hot Start Dna Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kod hot start dna polymerase/product/Millipore
    Average 99 stars, based on 221 article reviews
    Price from $9.99 to $1999.99
    kod hot start dna polymerase - by Bioz Stars, 2020-04
    99/100 stars
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    Performance of four thermostable DNA polymerases for the synthesis of gene B using PCA-DTF. Lane 1: negative control reaction performed at the same conditions, without addition of primers; lane 2: KOD Hot Start DNA polymerase; lane 3: Q5 Hot Start High Fidelity DNA polymerase; lane 4: Pfu Turbo DNA polymerase and lane 5: Taq DNA polymerase. M2: NZYladder I

    Journal: BMC Biotechnology

    Article Title: Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins

    doi: 10.1186/s12896-016-0316-3

    Figure Lengend Snippet: Performance of four thermostable DNA polymerases for the synthesis of gene B using PCA-DTF. Lane 1: negative control reaction performed at the same conditions, without addition of primers; lane 2: KOD Hot Start DNA polymerase; lane 3: Q5 Hot Start High Fidelity DNA polymerase; lane 4: Pfu Turbo DNA polymerase and lane 5: Taq DNA polymerase. M2: NZYladder I

    Article Snippet: Annealing occurred at 60 °C for 10 s for KOD Hot Start DNA polymerase and 60 °C for 30 s for Q5® Hot Star High Fidelity DNA polymerase, Pfu Turbo and Taq.

    Techniques: Negative Control

    A safe, programmable system to test CRISPR-based gene editing in haploid yeast. (A) Our design for a yeast system for analysis of CRISPR editing includes (i) an inducible S. pyogenes Cas9 expressed from a URA3 -based plasmid, (ii) a sgRNA expression cassette on a high-copy LEU2 -based plasmid, and (iii) a programmable gene “target” (consisting of a drug resistance marker cassette) at a safe-harbor locus ( HIS3 ) flanked by two “unique” DNA sequences (u1) that do not exist within the S. cerevisiae ). Induction of Cas9 allows targeting and double-stranded break formation at the identical (u1) sequences. In the absence of exogenous DNA ( e.g. ) to the flanking HIS3 UTR. The expected product sizes of the amplified PCR fragments are ∼379 bp (depending on the type of insertion/deletion(s) at the cut site, if any), or 1839 bp in the absence of any editing. Colonies were tested for resistance on medium containing G418 (below). (D) Clonal isolates from Cas9 editing (a dozen independent experiments) using the high copy sgRNA(u1) plasmid from (B) and that had also excised the selection cassette were analyzed by DNA sequencing at the HIS3 locus. The number of each genotype obtained is listed (right).

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae

    doi: 10.1534/g3.117.300557

    Figure Lengend Snippet: A safe, programmable system to test CRISPR-based gene editing in haploid yeast. (A) Our design for a yeast system for analysis of CRISPR editing includes (i) an inducible S. pyogenes Cas9 expressed from a URA3 -based plasmid, (ii) a sgRNA expression cassette on a high-copy LEU2 -based plasmid, and (iii) a programmable gene “target” (consisting of a drug resistance marker cassette) at a safe-harbor locus ( HIS3 ) flanked by two “unique” DNA sequences (u1) that do not exist within the S. cerevisiae ). Induction of Cas9 allows targeting and double-stranded break formation at the identical (u1) sequences. In the absence of exogenous DNA ( e.g. ) to the flanking HIS3 UTR. The expected product sizes of the amplified PCR fragments are ∼379 bp (depending on the type of insertion/deletion(s) at the cut site, if any), or 1839 bp in the absence of any editing. Colonies were tested for resistance on medium containing G418 (below). (D) Clonal isolates from Cas9 editing (a dozen independent experiments) using the high copy sgRNA(u1) plasmid from (B) and that had also excised the selection cassette were analyzed by DNA sequencing at the HIS3 locus. The number of each genotype obtained is listed (right).

    Article Snippet: Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore).

    Techniques: CRISPR, Plasmid Preparation, Expressing, Marker, Amplification, Polymerase Chain Reaction, Selection, DNA Sequencing

    Altering levels of Cas9 to activate an artificial gene drive in diploid yeast cells. (A) Our design of a programmable gene drive included (i) an integrated copy of S. pyogenes Cas9 (asterisk denotes use of various Cas9 fusions in an otherwise identical construct) under the inducible GAL1/10 promoter at the HIS3 locus in MAT ), and (iv) an artificial gene “target” containing a different selectable marker ( S. pombe HIS5 ) and flanked by (u1) artificial Cas9 sites at the HIS3 locus in a strain of the opposite mating type ( MAT α). (B) Activation and testing of all gene drives was performed as follows. First, the Cas9-containing strain (shown, GFY-2383) was transformed with the sgRNA(u1) plasmid (pGF-IVL1220) or an empty vector (pRS425) control and maintained on dextrose. Second, the gene drive strain ( MAT a) harboring the sgRNA(u1) plasmid was mated to the target strain ( MAT α; GFY-3206 or GFY-3207) on rich medium for 24 hr at 30°. Third, diploid yeast were selected twice on SD-LEU-HIS medium (24 hr incubation at 30°). Fourth, diploids were cultured overnight in S-LEU + Raffinose/Sucrose liquid medium. Fifth, strains were back-diluted to an OD 600 of ∼0.35 OD/ml in YP + Galactose and grown at 30° for various amounts of time. Sixth, yeast were harvested by a brief centrifugation, washed with water, diluted to ∼1000 cells/ml, and 0.5 ml was plated onto SD-LEU medium and incubated at 30° for 2 d. Finally, yeast were transferred by replica-plating to SD-LEU and SD-HIS plates and incubated for 24 additional hours before imaging. Representative plates are shown for the GFY-3206 cross. (C) Quantification of the percentage of colonies displaying an active gene drive (assayed by sensitivity on SD-HIS medium). Error, SD. Statistically significant comparisons are denoted using an unpaired t -test. N.S., not significant. The value for 0 hr is 0% drive activity, not 50%. Experimental runs with an empty plasmid (pRS425) were also performed and displayed a value of zero drive activity for all time points. (D) Clonal isolates were randomly selected from SD-LEU plates from (B) and retested on G418 and SD-HIS media. Multiple crosses were used to determine ploidy status. Diagnostic PCRs (A–D) were performed on isolated diploid chromosomal DNA to assess the HIS3 .

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae

    doi: 10.1534/g3.117.300557

    Figure Lengend Snippet: Altering levels of Cas9 to activate an artificial gene drive in diploid yeast cells. (A) Our design of a programmable gene drive included (i) an integrated copy of S. pyogenes Cas9 (asterisk denotes use of various Cas9 fusions in an otherwise identical construct) under the inducible GAL1/10 promoter at the HIS3 locus in MAT ), and (iv) an artificial gene “target” containing a different selectable marker ( S. pombe HIS5 ) and flanked by (u1) artificial Cas9 sites at the HIS3 locus in a strain of the opposite mating type ( MAT α). (B) Activation and testing of all gene drives was performed as follows. First, the Cas9-containing strain (shown, GFY-2383) was transformed with the sgRNA(u1) plasmid (pGF-IVL1220) or an empty vector (pRS425) control and maintained on dextrose. Second, the gene drive strain ( MAT a) harboring the sgRNA(u1) plasmid was mated to the target strain ( MAT α; GFY-3206 or GFY-3207) on rich medium for 24 hr at 30°. Third, diploid yeast were selected twice on SD-LEU-HIS medium (24 hr incubation at 30°). Fourth, diploids were cultured overnight in S-LEU + Raffinose/Sucrose liquid medium. Fifth, strains were back-diluted to an OD 600 of ∼0.35 OD/ml in YP + Galactose and grown at 30° for various amounts of time. Sixth, yeast were harvested by a brief centrifugation, washed with water, diluted to ∼1000 cells/ml, and 0.5 ml was plated onto SD-LEU medium and incubated at 30° for 2 d. Finally, yeast were transferred by replica-plating to SD-LEU and SD-HIS plates and incubated for 24 additional hours before imaging. Representative plates are shown for the GFY-3206 cross. (C) Quantification of the percentage of colonies displaying an active gene drive (assayed by sensitivity on SD-HIS medium). Error, SD. Statistically significant comparisons are denoted using an unpaired t -test. N.S., not significant. The value for 0 hr is 0% drive activity, not 50%. Experimental runs with an empty plasmid (pRS425) were also performed and displayed a value of zero drive activity for all time points. (D) Clonal isolates were randomly selected from SD-LEU plates from (B) and retested on G418 and SD-HIS media. Multiple crosses were used to determine ploidy status. Diagnostic PCRs (A–D) were performed on isolated diploid chromosomal DNA to assess the HIS3 .

    Article Snippet: Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore).

    Techniques: Construct, Marker, Activation Assay, Transformation Assay, Plasmid Preparation, Incubation, Cell Culture, Centrifugation, Imaging, Activity Assay, Diagnostic Assay, Isolation

    Altering levels of Cas9 to activate an artificial gene drive in diploid yeast cells. (A) Our design of a programmable gene drive included (i) an integrated copy of S. pyogenes Cas9 (asterisk denotes use of various Cas9 fusions in an otherwise identical construct) under the inducible GAL1/10 promoter at the HIS3 locus in MAT a cells, (ii) a Kanamycin-resistance gene cassette, (iii) flanking unique sites (u2) ( Finnigan and Thorner 2016 ) surrounding the entire gene drive system to be used as a genetic failsafe (see Figure S6 in File S1 ), and (iv) an artificial gene “target” containing a different selectable marker ( S. pombe HIS5 ) and flanked by (u1) artificial Cas9 sites at the HIS3 locus in a strain of the opposite mating type ( MAT α). (B) Activation and testing of all gene drives was performed as follows. First, the Cas9-containing strain (shown, GFY-2383) was transformed with the sgRNA(u1) plasmid (pGF-IVL1220) or an empty vector (pRS425) control and maintained on dextrose. Second, the gene drive strain ( MAT a) harboring the sgRNA(u1) plasmid was mated to the target strain ( MAT α; GFY-3206 or GFY-3207) on rich medium for 24 hr at 30°. Third, diploid yeast were selected twice on SD-LEU-HIS medium (24 hr incubation at 30°). Fourth, diploids were cultured overnight in S-LEU + Raffinose/Sucrose liquid medium. Fifth, strains were back-diluted to an OD 600 of ∼0.35 OD/ml in YP + Galactose and grown at 30° for various amounts of time. Sixth, yeast were harvested by a brief centrifugation, washed with water, diluted to ∼1000 cells/ml, and 0.5 ml was plated onto SD-LEU medium and incubated at 30° for 2 d. Finally, yeast were transferred by replica-plating to SD-LEU and SD-HIS plates and incubated for 24 additional hours before imaging. Representative plates are shown for the GFY-3206 cross. (C) Quantification of the percentage of colonies displaying an active gene drive (assayed by sensitivity on SD-HIS medium). Error, SD. Statistically significant comparisons are denoted using an unpaired t -test. N.S., not significant. The value for 0 hr is 0% drive activity, not 50%. Experimental runs with an empty plasmid (pRS425) were also performed and displayed a value of zero drive activity for all time points. (D) Clonal isolates were randomly selected from SD-LEU plates from (B) and retested on G418 and SD-HIS media. Multiple crosses were used to determine ploidy status. Diagnostic PCRs (A–D) were performed on isolated diploid chromosomal DNA to assess the HIS3 locus at 0 and 12 hr post galactose shift (also see Figure S4 in File S1 ). Oligonucleotides used can be found in Table S1 in File S1 .

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae

    doi: 10.1534/g3.117.300557

    Figure Lengend Snippet: Altering levels of Cas9 to activate an artificial gene drive in diploid yeast cells. (A) Our design of a programmable gene drive included (i) an integrated copy of S. pyogenes Cas9 (asterisk denotes use of various Cas9 fusions in an otherwise identical construct) under the inducible GAL1/10 promoter at the HIS3 locus in MAT a cells, (ii) a Kanamycin-resistance gene cassette, (iii) flanking unique sites (u2) ( Finnigan and Thorner 2016 ) surrounding the entire gene drive system to be used as a genetic failsafe (see Figure S6 in File S1 ), and (iv) an artificial gene “target” containing a different selectable marker ( S. pombe HIS5 ) and flanked by (u1) artificial Cas9 sites at the HIS3 locus in a strain of the opposite mating type ( MAT α). (B) Activation and testing of all gene drives was performed as follows. First, the Cas9-containing strain (shown, GFY-2383) was transformed with the sgRNA(u1) plasmid (pGF-IVL1220) or an empty vector (pRS425) control and maintained on dextrose. Second, the gene drive strain ( MAT a) harboring the sgRNA(u1) plasmid was mated to the target strain ( MAT α; GFY-3206 or GFY-3207) on rich medium for 24 hr at 30°. Third, diploid yeast were selected twice on SD-LEU-HIS medium (24 hr incubation at 30°). Fourth, diploids were cultured overnight in S-LEU + Raffinose/Sucrose liquid medium. Fifth, strains were back-diluted to an OD 600 of ∼0.35 OD/ml in YP + Galactose and grown at 30° for various amounts of time. Sixth, yeast were harvested by a brief centrifugation, washed with water, diluted to ∼1000 cells/ml, and 0.5 ml was plated onto SD-LEU medium and incubated at 30° for 2 d. Finally, yeast were transferred by replica-plating to SD-LEU and SD-HIS plates and incubated for 24 additional hours before imaging. Representative plates are shown for the GFY-3206 cross. (C) Quantification of the percentage of colonies displaying an active gene drive (assayed by sensitivity on SD-HIS medium). Error, SD. Statistically significant comparisons are denoted using an unpaired t -test. N.S., not significant. The value for 0 hr is 0% drive activity, not 50%. Experimental runs with an empty plasmid (pRS425) were also performed and displayed a value of zero drive activity for all time points. (D) Clonal isolates were randomly selected from SD-LEU plates from (B) and retested on G418 and SD-HIS media. Multiple crosses were used to determine ploidy status. Diagnostic PCRs (A–D) were performed on isolated diploid chromosomal DNA to assess the HIS3 locus at 0 and 12 hr post galactose shift (also see Figure S4 in File S1 ). Oligonucleotides used can be found in Table S1 in File S1 .

    Article Snippet: Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore).

    Techniques: Construct, Marker, Activation Assay, Transformation Assay, Plasmid Preparation, Incubation, Cell Culture, Centrifugation, Imaging, Activity Assay, Diagnostic Assay, Isolation

    A safe, programmable system to test CRISPR-based gene editing in haploid yeast. (A) Our design for a yeast system for analysis of CRISPR editing includes (i) an inducible S. pyogenes Cas9 expressed from a URA3 -based plasmid, (ii) a sgRNA expression cassette on a high-copy LEU2 -based plasmid, and (iii) a programmable gene “target” (consisting of a drug resistance marker cassette) at a safe-harbor locus ( HIS3 ) flanked by two “unique” DNA sequences (u1) that do not exist within the S. cerevisiae genome ( Finnigan and Thorner 2016 ). Induction of Cas9 allows targeting and double-stranded break formation at the identical (u1) sequences. In the absence of exogenous DNA ( e.g. , amplified PCR product) to be used for HDR, NHEJ of the exposed chromosomal ends causes full excision of the selectable marker. However, given the unique arrangement of the identical (u1) sites, NHEJ in the absence of any insertion/deletion mutation at the Cas9 cut site (left) recreates another WT (u1) site and subsequent re-editing of the same target sequence until Cas9 expression is shutoff or a mutation is positioned within the (u1) site (right). (B) Cas9-dependent editing results in cell inviability. GFY-2353 yeast already harboring Cas9-NLS on a vector (pGF-IVL1116) or an empty vector control (pRS316) were induced in medium containing galactose, transformed with the sgRNA(u1)-expression cassette on either a CEN-based (pGF-V1215) or 2μ-based (pGF-V1220) plasmid, and plated onto SD-URA-LEU media. (C) GFY-2588 yeast containing pGF-IVL1342 were transformed with sgRNA(u1) plasmid (pGF-V1216) and selected on SD-URA-LEU medium. The isolated chromosomal DNA of individual clonal (surviving) isolates was assayed by PCR using DNA oligonucleotides (F1/R1, Table S1 in File S1 ) to the flanking HIS3 UTR. The expected product sizes of the amplified PCR fragments are ∼379 bp (depending on the type of insertion/deletion(s) at the cut site, if any), or 1839 bp in the absence of any editing. Colonies were tested for resistance on medium containing G418 (below). (D) Clonal isolates from Cas9 editing (a dozen independent experiments) using the high copy sgRNA(u1) plasmid from (B) and that had also excised the selection cassette were analyzed by DNA sequencing at the HIS3 locus. The number of each genotype obtained is listed (right).

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae

    doi: 10.1534/g3.117.300557

    Figure Lengend Snippet: A safe, programmable system to test CRISPR-based gene editing in haploid yeast. (A) Our design for a yeast system for analysis of CRISPR editing includes (i) an inducible S. pyogenes Cas9 expressed from a URA3 -based plasmid, (ii) a sgRNA expression cassette on a high-copy LEU2 -based plasmid, and (iii) a programmable gene “target” (consisting of a drug resistance marker cassette) at a safe-harbor locus ( HIS3 ) flanked by two “unique” DNA sequences (u1) that do not exist within the S. cerevisiae genome ( Finnigan and Thorner 2016 ). Induction of Cas9 allows targeting and double-stranded break formation at the identical (u1) sequences. In the absence of exogenous DNA ( e.g. , amplified PCR product) to be used for HDR, NHEJ of the exposed chromosomal ends causes full excision of the selectable marker. However, given the unique arrangement of the identical (u1) sites, NHEJ in the absence of any insertion/deletion mutation at the Cas9 cut site (left) recreates another WT (u1) site and subsequent re-editing of the same target sequence until Cas9 expression is shutoff or a mutation is positioned within the (u1) site (right). (B) Cas9-dependent editing results in cell inviability. GFY-2353 yeast already harboring Cas9-NLS on a vector (pGF-IVL1116) or an empty vector control (pRS316) were induced in medium containing galactose, transformed with the sgRNA(u1)-expression cassette on either a CEN-based (pGF-V1215) or 2μ-based (pGF-V1220) plasmid, and plated onto SD-URA-LEU media. (C) GFY-2588 yeast containing pGF-IVL1342 were transformed with sgRNA(u1) plasmid (pGF-V1216) and selected on SD-URA-LEU medium. The isolated chromosomal DNA of individual clonal (surviving) isolates was assayed by PCR using DNA oligonucleotides (F1/R1, Table S1 in File S1 ) to the flanking HIS3 UTR. The expected product sizes of the amplified PCR fragments are ∼379 bp (depending on the type of insertion/deletion(s) at the cut site, if any), or 1839 bp in the absence of any editing. Colonies were tested for resistance on medium containing G418 (below). (D) Clonal isolates from Cas9 editing (a dozen independent experiments) using the high copy sgRNA(u1) plasmid from (B) and that had also excised the selection cassette were analyzed by DNA sequencing at the HIS3 locus. The number of each genotype obtained is listed (right).

    Article Snippet: Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore).

    Techniques: CRISPR, Plasmid Preparation, Expressing, Marker, Amplification, Polymerase Chain Reaction, Non-Homologous End Joining, Mutagenesis, Sequencing, Transformation Assay, Isolation, Selection, DNA Sequencing