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TaKaRa polymerase chain reaction pcr buffer
Polymerase Chain Reaction Pcr Buffer, supplied by TaKaRa, used in various techniques. Bioz Stars score: 89/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/polymerase chain reaction pcr buffer/product/TaKaRa
Average 89 stars, based on 5 article reviews
Price from $9.99 to $1999.99
polymerase chain reaction pcr buffer - by Bioz Stars, 2020-07
89/100 stars

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SYBR Green Assay:

Article Title: Folic acid reverses uric acid crystal-induced surface OAT1 internalization by inhibiting RhoA activity in uric acid nephropathy
Article Snippet: .. TRIzol, reverse transcription buffer, SYBR green I polymerase chain reaction (PCR) buffer, dNTPs, MMLV and Taq DNA polymerase were purchased from Takara Biotechnology, Co., Ltd. (Dalian, China). .. Rabbit polyclonal OAT1 antibody purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Polymerase Chain Reaction:

Article Title: Folic acid reverses uric acid crystal-induced surface OAT1 internalization by inhibiting RhoA activity in uric acid nephropathy
Article Snippet: .. TRIzol, reverse transcription buffer, SYBR green I polymerase chain reaction (PCR) buffer, dNTPs, MMLV and Taq DNA polymerase were purchased from Takara Biotechnology, Co., Ltd. (Dalian, China). .. Rabbit polyclonal OAT1 antibody purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Article Title: Paracrine effects of transplanted mesothelial cells isolated from temperature-sensitive SV40 large T-antigen gene transgenic rats during peritoneal repair
Article Snippet: .. The product was added to 2 μL of 10× polymerase chain reaction (PCR) buffer (Takara Bio, Inc., Shiga, Japan), 1 μL of Protector RNase Inhibitor (Roche Diagnostics Corp., Mannheim, Germany) and 0.5 μL of M-MLV Reverse Transcriptase (Invitrogen Corp., Carlsbad, CA, USA), followed by incubation at 42°C for 60 min. .. The complementary DNA (cDNA) product was used for real-time PCR.

Article Title: Precocious genotypes and homozygous tendency generated by self-pollination in walnut
Article Snippet: .. The amplification reaction was performed in a volume of 20 μL containing 1× polymerase chain reaction (PCR) buffer, 50 ng of genomic DNA, 300 μM dNTPs, 0.4 μM of each primer, 3 mM of MgCl2 , and 1 unit of Taq DNA polymerase (TaKaRa Biotechnology, Dalian, China). .. PCR was performed on an ABI GeneAmp®9700 thermal cycler (Applied Biosystems, Foster City, CA, USA) with the following program: an initial 5-min incubation at 94 °C, 35 cycles of 45 s at 94 °C, 45 s at the annealing temperature of 50–55 °C, 45 s at 72 °C, and a final incubation at 72 °C for 5 min. After amplification, 3 μL of each sample were separated in a 6% denaturing polyacrylamide gel with 7 M urea and 1 × TBE (Tris-borate-EDTA) buffer, and visualized by silver staining.

Incubation:

Article Title: Paracrine effects of transplanted mesothelial cells isolated from temperature-sensitive SV40 large T-antigen gene transgenic rats during peritoneal repair
Article Snippet: .. The product was added to 2 μL of 10× polymerase chain reaction (PCR) buffer (Takara Bio, Inc., Shiga, Japan), 1 μL of Protector RNase Inhibitor (Roche Diagnostics Corp., Mannheim, Germany) and 0.5 μL of M-MLV Reverse Transcriptase (Invitrogen Corp., Carlsbad, CA, USA), followed by incubation at 42°C for 60 min. .. The complementary DNA (cDNA) product was used for real-time PCR.

Amplification:

Article Title: Precocious genotypes and homozygous tendency generated by self-pollination in walnut
Article Snippet: .. The amplification reaction was performed in a volume of 20 μL containing 1× polymerase chain reaction (PCR) buffer, 50 ng of genomic DNA, 300 μM dNTPs, 0.4 μM of each primer, 3 mM of MgCl2 , and 1 unit of Taq DNA polymerase (TaKaRa Biotechnology, Dalian, China). .. PCR was performed on an ABI GeneAmp®9700 thermal cycler (Applied Biosystems, Foster City, CA, USA) with the following program: an initial 5-min incubation at 94 °C, 35 cycles of 45 s at 94 °C, 45 s at the annealing temperature of 50–55 °C, 45 s at 72 °C, and a final incubation at 72 °C for 5 min. After amplification, 3 μL of each sample were separated in a 6% denaturing polyacrylamide gel with 7 M urea and 1 × TBE (Tris-borate-EDTA) buffer, and visualized by silver staining.

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  • 95
    TaKaRa pcr buffer
    Effect of <t>DNA</t> methylation inhibitor and DNMT1 gene knockdown on SOCS gene expression. CaSki (A,D), HeLa (B,E), and ME-180 (C,F) cells were treated with 10 μM of 5-Azacytosine (5-Aza) for 72 h (A-C) or infected with control lentivirus (shSCR) or lentivirus expressing shRNA targeting DNMT1 (shDNMT1) (D-F). Knock-down of DNMT1 and SOCS gene expression was examined with <t>qRT-PCR.</t> Asterisk (*), statistically significant ( p
    Pcr Buffer, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1840 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr buffer/product/TaKaRa
    Average 95 stars, based on 1840 article reviews
    Price from $9.99 to $1999.99
    pcr buffer - by Bioz Stars, 2020-07
    95/100 stars
      Buy from Supplier

    94
    TaKaRa pcr reaction buffer
    Ct values and copy number of <t>HBV</t> serum samples originated from three standard curves corresponding to the S (A), C (B) and X (C) regions respectively. Each real-time <t>PCR</t> reaction was performed in triplicate.
    Pcr Reaction Buffer, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr reaction buffer/product/TaKaRa
    Average 94 stars, based on 89 article reviews
    Price from $9.99 to $1999.99
    pcr reaction buffer - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    92
    TaKaRa m8 egfp bac stock solution
    Growth of the recovered virus from <t>pLC16m8-BAC</t> named vLC16m8-BAC. RK13 cells were infected with either authentic m8, <t>m8-EGFP-BAC,</t> or vLC16m8-BAC at an MOI of 0.01. The infected cells along with the culture media at the indicated d.p.i. were collected and freeze-thawed. The amount of the virus was determined by a standard plaque assay.
    M8 Egfp Bac Stock Solution, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m8 egfp bac stock solution/product/TaKaRa
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    m8 egfp bac stock solution - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

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    Effect of DNA methylation inhibitor and DNMT1 gene knockdown on SOCS gene expression. CaSki (A,D), HeLa (B,E), and ME-180 (C,F) cells were treated with 10 μM of 5-Azacytosine (5-Aza) for 72 h (A-C) or infected with control lentivirus (shSCR) or lentivirus expressing shRNA targeting DNMT1 (shDNMT1) (D-F). Knock-down of DNMT1 and SOCS gene expression was examined with qRT-PCR. Asterisk (*), statistically significant ( p

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling (SOCS) Genes Are Silenced by DNA Hypermethylation and Histone Deacetylation and Regulate Response to Radiotherapy in Cervical Cancer Cells

    doi: 10.1371/journal.pone.0123133

    Figure Lengend Snippet: Effect of DNA methylation inhibitor and DNMT1 gene knockdown on SOCS gene expression. CaSki (A,D), HeLa (B,E), and ME-180 (C,F) cells were treated with 10 μM of 5-Azacytosine (5-Aza) for 72 h (A-C) or infected with control lentivirus (shSCR) or lentivirus expressing shRNA targeting DNMT1 (shDNMT1) (D-F). Knock-down of DNMT1 and SOCS gene expression was examined with qRT-PCR. Asterisk (*), statistically significant ( p

    Article Snippet: Briefly, a 20 μL reaction volume containing 25 ng bisulfite modified DNA, 1× PCR buffer, 1.5 mM MgCl2 , 0.25 mM dNTPs, 0.5 μM specific primer mix and 1 unit Ex-Taq Hot Start enzyme (Takara BioInc) was used.

    Techniques: DNA Methylation Assay, Expressing, Infection, shRNA, Quantitative RT-PCR

    DNA methylation analysis of SOCS gene promoter. (A) Methyl-specific PCR analysis. Bisulfite-treated genomic DNA was amplified with unmethylated (U) or methylated (M) DNA specific primers. (B-D) Bisulfite sequencing of SOCS1 (B), SOCS3 (C), and SOCS5 (D). Unmethylated CpG site in amplified promoter region was showed as an open circle and methylated CpG as a closed circle.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling (SOCS) Genes Are Silenced by DNA Hypermethylation and Histone Deacetylation and Regulate Response to Radiotherapy in Cervical Cancer Cells

    doi: 10.1371/journal.pone.0123133

    Figure Lengend Snippet: DNA methylation analysis of SOCS gene promoter. (A) Methyl-specific PCR analysis. Bisulfite-treated genomic DNA was amplified with unmethylated (U) or methylated (M) DNA specific primers. (B-D) Bisulfite sequencing of SOCS1 (B), SOCS3 (C), and SOCS5 (D). Unmethylated CpG site in amplified promoter region was showed as an open circle and methylated CpG as a closed circle.

    Article Snippet: Briefly, a 20 μL reaction volume containing 25 ng bisulfite modified DNA, 1× PCR buffer, 1.5 mM MgCl2 , 0.25 mM dNTPs, 0.5 μM specific primer mix and 1 unit Ex-Taq Hot Start enzyme (Takara BioInc) was used.

    Techniques: DNA Methylation Assay, Polymerase Chain Reaction, Amplification, Methylation, Methylation Sequencing

    Heterogeneous expression of CD14 mRNA by CD14 high and CD14 low HGF. CD14 high HGF (lane 1, donor MH; lane 2, donor SM) and CD14 low HGF (lane 3, donor KT; lane 4, donor NK) from confluent cultures were collected by trypsinization. RNA was extracted from the cells, and its cDNA was prepared and analyzed by RT-PCR. The results are representative of three different experiments.

    Journal: Infection and Immunity

    Article Title: Heterogeneous Expression and Release of CD14 by Human Gingival Fibroblasts: Characterization and CD14-Mediated Interleukin-8 Secretion in Response to Lipopolysaccharide

    doi:

    Figure Lengend Snippet: Heterogeneous expression of CD14 mRNA by CD14 high and CD14 low HGF. CD14 high HGF (lane 1, donor MH; lane 2, donor SM) and CD14 low HGF (lane 3, donor KT; lane 4, donor NK) from confluent cultures were collected by trypsinization. RNA was extracted from the cells, and its cDNA was prepared and analyzed by RT-PCR. The results are representative of three different experiments.

    Article Snippet: The PCR mixture contained 5 μl of the cDNA mixture, 2 μl of 10× PCR buffer, 0.2 mM deoxynucleoside triphosphates, 50 pmol of each primer, and 0.1 μl of Ex Taq DNA polymerase (Takara, Tokyo, Japan) in a total volume of 20 μl.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Ct values and copy number of HBV serum samples originated from three standard curves corresponding to the S (A), C (B) and X (C) regions respectively. Each real-time PCR reaction was performed in triplicate.

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA

    doi: 10.3748/wjg.v12.i45.7365

    Figure Lengend Snippet: Ct values and copy number of HBV serum samples originated from three standard curves corresponding to the S (A), C (B) and X (C) regions respectively. Each real-time PCR reaction was performed in triplicate.

    Article Snippet: Real-time PCR amplification was performed in 20 μL reaction mixture containing 1 μL standard DNA or 2 μL isolated serum HBV DNA, 2 × PCR reaction buffer [100 mmol/L Tris-HCl (pH8.3), 100 mmol/L KCl, 7.0 mmol/L MgCl2 , 400 μmol/L each of the deoxynucleotide triphosphates (dNTP), 1U hot star DNA polymerase Takara, Japan], 5pmol of each pair of primers and 2.5 pmol of corresponding TaqMan probe.

    Techniques: Real-time Polymerase Chain Reaction

    Quantitation of HBV viral DNA in sera by real-time PCR

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA

    doi: 10.3748/wjg.v12.i45.7365

    Figure Lengend Snippet: Quantitation of HBV viral DNA in sera by real-time PCR

    Article Snippet: Real-time PCR amplification was performed in 20 μL reaction mixture containing 1 μL standard DNA or 2 μL isolated serum HBV DNA, 2 × PCR reaction buffer [100 mmol/L Tris-HCl (pH8.3), 100 mmol/L KCl, 7.0 mmol/L MgCl2 , 400 μmol/L each of the deoxynucleotide triphosphates (dNTP), 1U hot star DNA polymerase Takara, Japan], 5pmol of each pair of primers and 2.5 pmol of corresponding TaqMan probe.

    Techniques: Quantitation Assay, Real-time Polymerase Chain Reaction

    Growth of the recovered virus from pLC16m8-BAC named vLC16m8-BAC. RK13 cells were infected with either authentic m8, m8-EGFP-BAC, or vLC16m8-BAC at an MOI of 0.01. The infected cells along with the culture media at the indicated d.p.i. were collected and freeze-thawed. The amount of the virus was determined by a standard plaque assay.

    Journal: PLoS ONE

    Article Title: Construction and characterization of bacterial artificial chromosomes harboring the full-length genome of a highly attenuated vaccinia virus LC16m8

    doi: 10.1371/journal.pone.0192725

    Figure Lengend Snippet: Growth of the recovered virus from pLC16m8-BAC named vLC16m8-BAC. RK13 cells were infected with either authentic m8, m8-EGFP-BAC, or vLC16m8-BAC at an MOI of 0.01. The infected cells along with the culture media at the indicated d.p.i. were collected and freeze-thawed. The amount of the virus was determined by a standard plaque assay.

    Article Snippet: First, 8.5 μl of m8-EGFP-BAC stock solution (approximately 106 to 107 pfu/ml of viruses in DMEM-5 FBS) was mixed with 1 μl of 10 x Proteinase K lysis buffer (100 mM Tris-Cl; pH 8.0, 4.5% NP40, and 4.5% Tween 20) and 0.5 μl of 20 mg/ml proteinase K (Takara, Shiga, Japan).

    Techniques: BAC Assay, Infection, Plaque Assay

    Identity of the ITR region in authentic m8 and 8.8S-clone4, a clone that was plaque-purified from EGFP-negative and bacterial replicon-free vLC16m8.8S-BAC. The coverage depth computed by the alignment of short-reads to the sequence of the left ITR region, which was extracted from the consensus sequence of pLC16m8.8S-BAC is shown (A and D). The coverage depth in the ITR left region from the terminus (position 1) to the C23L gene of m8 or 8.8S-clone 4 was plotted as either a black or a red line. The mean of the coverage depth by each of the regions (i.e., 70 base pair repeat, non-repeated region II to 125 base pairs, 54 base pair repeat, non-repeated region III, and C23L) was plotted (B, C, E, and F). One-way ANOVA with Bonferroni's multiple-comparison test was used to determine the level of statistical significance. The effect size (r), which indicates the magnitude of the mean difference between the regions, was also calculated. The mean of coverage depth at the 70 base pairs repeats were calculated from position 474 to 1423 to exclude the effect of low coverage depth artificially occurred at the terminus of the reference sequence. The number of 54 base pair tandem repeats in the reference sequence was artificially modified from x 41 (A-C) to x 23 (D-E). “n.s.” indicates

    Journal: PLoS ONE

    Article Title: Construction and characterization of bacterial artificial chromosomes harboring the full-length genome of a highly attenuated vaccinia virus LC16m8

    doi: 10.1371/journal.pone.0192725

    Figure Lengend Snippet: Identity of the ITR region in authentic m8 and 8.8S-clone4, a clone that was plaque-purified from EGFP-negative and bacterial replicon-free vLC16m8.8S-BAC. The coverage depth computed by the alignment of short-reads to the sequence of the left ITR region, which was extracted from the consensus sequence of pLC16m8.8S-BAC is shown (A and D). The coverage depth in the ITR left region from the terminus (position 1) to the C23L gene of m8 or 8.8S-clone 4 was plotted as either a black or a red line. The mean of the coverage depth by each of the regions (i.e., 70 base pair repeat, non-repeated region II to 125 base pairs, 54 base pair repeat, non-repeated region III, and C23L) was plotted (B, C, E, and F). One-way ANOVA with Bonferroni's multiple-comparison test was used to determine the level of statistical significance. The effect size (r), which indicates the magnitude of the mean difference between the regions, was also calculated. The mean of coverage depth at the 70 base pairs repeats were calculated from position 474 to 1423 to exclude the effect of low coverage depth artificially occurred at the terminus of the reference sequence. The number of 54 base pair tandem repeats in the reference sequence was artificially modified from x 41 (A-C) to x 23 (D-E). “n.s.” indicates "not significant”.

    Article Snippet: First, 8.5 μl of m8-EGFP-BAC stock solution (approximately 106 to 107 pfu/ml of viruses in DMEM-5 FBS) was mixed with 1 μl of 10 x Proteinase K lysis buffer (100 mM Tris-Cl; pH 8.0, 4.5% NP40, and 4.5% Tween 20) and 0.5 μl of 20 mg/ml proteinase K (Takara, Shiga, Japan).

    Techniques: Purification, BAC Assay, Sequencing, Modification

    Generation of the m8-BAC system, which enables recovery of the bacterial replicon-free m8. The scheme to modify pLC16m8-BAC to pLC16m8.8S/8.OS-BAC and then to pLC16m8.8/8.O-BAC is shown. The first recombination was performed to insert PCR products of I-SceI site and a gene expression cassette conferred kanamycin resistance (Kan R ) with the rear part of B5R (B5R-R) harbored by flanks homologous to parts of B5R-F and EGFP (α and β in the rectangles) cassette into pLC16m8-BAC (A). The flank α contained either of the nucleotide sequences that resulted defective (m8 type) or functional (mO type) B5R gene product. The second red recombination, which was achieved by induction of I-SceI digestion system with the addition of L-arabinose was performed to make both pLC16m8.8S-BAC and pLC16m8.OS-BAC by excision of the Kan R . Either m8 type or mO type B5R gene product was expressed, when the virus was recovered from either the pLC16m8.8S-BAC or pLC16m8.OS-BAC, and the EGFP and mini-F cassettes are expected to be self-excised by the homologous recombination while recovering the viruses (B). pLC16m8.8-BAC and pLC16m8.O-BAC were made from pLC16m8.8S-BAC and pLC16m8.OS-BAC, respectively (C). The first Recombination was performed to remove the B5R-R from either pLC16m8.8S-BAC or pLC16m8.OS-BAC by substitution of a PCR product. The PCR product harbored a gene expression cassette conferred zeocin resistance (Zeo R ), which was flanked by homologous to part of B5R-R and the untranslated region besides B5R-R (γ and δ in the rectangles). Both pLC16m8.8-BAC and pLC16m8.O-BAC were made by excision of the Zeo R by the second red recombination. The virus recovered from pLC16m8.8-BAC or pLC16m8.O-BAC is expected to stably harbor the EGFP and mini-F cassettes. The target region of primer A, B, and C used for following the PCR are indicated.

    Journal: PLoS ONE

    Article Title: Construction and characterization of bacterial artificial chromosomes harboring the full-length genome of a highly attenuated vaccinia virus LC16m8

    doi: 10.1371/journal.pone.0192725

    Figure Lengend Snippet: Generation of the m8-BAC system, which enables recovery of the bacterial replicon-free m8. The scheme to modify pLC16m8-BAC to pLC16m8.8S/8.OS-BAC and then to pLC16m8.8/8.O-BAC is shown. The first recombination was performed to insert PCR products of I-SceI site and a gene expression cassette conferred kanamycin resistance (Kan R ) with the rear part of B5R (B5R-R) harbored by flanks homologous to parts of B5R-F and EGFP (α and β in the rectangles) cassette into pLC16m8-BAC (A). The flank α contained either of the nucleotide sequences that resulted defective (m8 type) or functional (mO type) B5R gene product. The second red recombination, which was achieved by induction of I-SceI digestion system with the addition of L-arabinose was performed to make both pLC16m8.8S-BAC and pLC16m8.OS-BAC by excision of the Kan R . Either m8 type or mO type B5R gene product was expressed, when the virus was recovered from either the pLC16m8.8S-BAC or pLC16m8.OS-BAC, and the EGFP and mini-F cassettes are expected to be self-excised by the homologous recombination while recovering the viruses (B). pLC16m8.8-BAC and pLC16m8.O-BAC were made from pLC16m8.8S-BAC and pLC16m8.OS-BAC, respectively (C). The first Recombination was performed to remove the B5R-R from either pLC16m8.8S-BAC or pLC16m8.OS-BAC by substitution of a PCR product. The PCR product harbored a gene expression cassette conferred zeocin resistance (Zeo R ), which was flanked by homologous to part of B5R-R and the untranslated region besides B5R-R (γ and δ in the rectangles). Both pLC16m8.8-BAC and pLC16m8.O-BAC were made by excision of the Zeo R by the second red recombination. The virus recovered from pLC16m8.8-BAC or pLC16m8.O-BAC is expected to stably harbor the EGFP and mini-F cassettes. The target region of primer A, B, and C used for following the PCR are indicated.

    Article Snippet: First, 8.5 μl of m8-EGFP-BAC stock solution (approximately 106 to 107 pfu/ml of viruses in DMEM-5 FBS) was mixed with 1 μl of 10 x Proteinase K lysis buffer (100 mM Tris-Cl; pH 8.0, 4.5% NP40, and 4.5% Tween 20) and 0.5 μl of 20 mg/ml proteinase K (Takara, Shiga, Japan).

    Techniques: BAC Assay, Polymerase Chain Reaction, Expressing, Functional Assay, Homologous Recombination, Stable Transfection

    Restriction fragment analysis of BAC plasmids. Prediction of NcoI digested fragment patterns based on the sequences of pLC16m8-BAC and the derivatives were shown (A). Purified BAC plasmids, pLC16m8-BAC (m8-BAC), pLC16m8.8S/8.OS-BAC (8.8S or 8.OS) and pLC16m8.8/8.O (8.8/8.O) were digested with NcoI and separated on a 0.75% agarose gel (B). Color of the gel was inverted. Sizes of a molecular weight marker are shown as marker, and the sizes are given. Changes in the restriction pattern due to the presence of EGFP, mini-F cassettes or rear part of B5R are indicated with a red arrow.

    Journal: PLoS ONE

    Article Title: Construction and characterization of bacterial artificial chromosomes harboring the full-length genome of a highly attenuated vaccinia virus LC16m8

    doi: 10.1371/journal.pone.0192725

    Figure Lengend Snippet: Restriction fragment analysis of BAC plasmids. Prediction of NcoI digested fragment patterns based on the sequences of pLC16m8-BAC and the derivatives were shown (A). Purified BAC plasmids, pLC16m8-BAC (m8-BAC), pLC16m8.8S/8.OS-BAC (8.8S or 8.OS) and pLC16m8.8/8.O (8.8/8.O) were digested with NcoI and separated on a 0.75% agarose gel (B). Color of the gel was inverted. Sizes of a molecular weight marker are shown as marker, and the sizes are given. Changes in the restriction pattern due to the presence of EGFP, mini-F cassettes or rear part of B5R are indicated with a red arrow.

    Article Snippet: First, 8.5 μl of m8-EGFP-BAC stock solution (approximately 106 to 107 pfu/ml of viruses in DMEM-5 FBS) was mixed with 1 μl of 10 x Proteinase K lysis buffer (100 mM Tris-Cl; pH 8.0, 4.5% NP40, and 4.5% Tween 20) and 0.5 μl of 20 mg/ml proteinase K (Takara, Shiga, Japan).

    Techniques: BAC Assay, Purification, Agarose Gel Electrophoresis, Molecular Weight, Marker

    Scheme to generate a VAC-BAC plasmid, pLC16m8-BAC. A recombinant VAC, m8-EGFP-BAC, was generated by homologous recombination accelerated by a CRISPR-Cas9 system (A). pVAC-BAC11 harbors an EGFP expression cassette driven by an early and late VAC promoter and a mini-F cassette, which is essential for plasmid maintenance in E . coli . The cassettes were flanked by the front (B5R-F) and rear (B5R-R) part of the VAC B5R gene. Note that the gRNA targeted the sequence on the m8 genome but not on the pVAC-BAC11. m8-EGFP-BAC was used to infect RK13 cells (B). The virus genome formed replication intermediates, concatemers, some of which were circularized was purified from the infected RK13 cells with the addition of IβT. An electrocompetent E . coli was transformed by a circularized virus genome (arrows) of the concatemerized genome, and the chloramphenicol-resistant cells were cloned (C). The VAC-BAC plasmids obtained from the clones were named pLC16m8-BAC.

    Journal: PLoS ONE

    Article Title: Construction and characterization of bacterial artificial chromosomes harboring the full-length genome of a highly attenuated vaccinia virus LC16m8

    doi: 10.1371/journal.pone.0192725

    Figure Lengend Snippet: Scheme to generate a VAC-BAC plasmid, pLC16m8-BAC. A recombinant VAC, m8-EGFP-BAC, was generated by homologous recombination accelerated by a CRISPR-Cas9 system (A). pVAC-BAC11 harbors an EGFP expression cassette driven by an early and late VAC promoter and a mini-F cassette, which is essential for plasmid maintenance in E . coli . The cassettes were flanked by the front (B5R-F) and rear (B5R-R) part of the VAC B5R gene. Note that the gRNA targeted the sequence on the m8 genome but not on the pVAC-BAC11. m8-EGFP-BAC was used to infect RK13 cells (B). The virus genome formed replication intermediates, concatemers, some of which were circularized was purified from the infected RK13 cells with the addition of IβT. An electrocompetent E . coli was transformed by a circularized virus genome (arrows) of the concatemerized genome, and the chloramphenicol-resistant cells were cloned (C). The VAC-BAC plasmids obtained from the clones were named pLC16m8-BAC.

    Article Snippet: First, 8.5 μl of m8-EGFP-BAC stock solution (approximately 106 to 107 pfu/ml of viruses in DMEM-5 FBS) was mixed with 1 μl of 10 x Proteinase K lysis buffer (100 mM Tris-Cl; pH 8.0, 4.5% NP40, and 4.5% Tween 20) and 0.5 μl of 20 mg/ml proteinase K (Takara, Shiga, Japan).

    Techniques: BAC Assay, Plasmid Preparation, Recombinant, Generated, Homologous Recombination, CRISPR, Expressing, Sequencing, Purification, Infection, Transformation Assay, Clone Assay

    Confirmation of self-excising EGFP and mini-F cassettes from the virus genome. The viral genomic DNA purified from the RK13 cells infected with either vLC16m8.O-BAC (crude viruses) (8.O), vLC16m8.8-BAC (crude viruses) (8.8), vLC16m8.OS-BAC (crude or an EGFP-negative plaque cloned viruses) (8.OS), or vLC16m8.8S-BAC (crude or an EGFP-negative plaque cloned viruses) (8.8S) was amplified by conventional PCR using either the primer set A/B or A/C. The target position of the primers is shown in Fig 3A and 3B . The no template control (NTC) monitors unintentional PCR amplification to produce a false-positive result.

    Journal: PLoS ONE

    Article Title: Construction and characterization of bacterial artificial chromosomes harboring the full-length genome of a highly attenuated vaccinia virus LC16m8

    doi: 10.1371/journal.pone.0192725

    Figure Lengend Snippet: Confirmation of self-excising EGFP and mini-F cassettes from the virus genome. The viral genomic DNA purified from the RK13 cells infected with either vLC16m8.O-BAC (crude viruses) (8.O), vLC16m8.8-BAC (crude viruses) (8.8), vLC16m8.OS-BAC (crude or an EGFP-negative plaque cloned viruses) (8.OS), or vLC16m8.8S-BAC (crude or an EGFP-negative plaque cloned viruses) (8.8S) was amplified by conventional PCR using either the primer set A/B or A/C. The target position of the primers is shown in Fig 3A and 3B . The no template control (NTC) monitors unintentional PCR amplification to produce a false-positive result.

    Article Snippet: First, 8.5 μl of m8-EGFP-BAC stock solution (approximately 106 to 107 pfu/ml of viruses in DMEM-5 FBS) was mixed with 1 μl of 10 x Proteinase K lysis buffer (100 mM Tris-Cl; pH 8.0, 4.5% NP40, and 4.5% Tween 20) and 0.5 μl of 20 mg/ml proteinase K (Takara, Shiga, Japan).

    Techniques: Purification, Infection, BAC Assay, Amplification, Polymerase Chain Reaction

    The characteristics of the viruses recovered from the pLC16m8.8S-BAC or pLC16m8.OS-BAC. The plaque size and EGFP expression were observed under phase-contrast and fluorescent microscopy. RK13 cells were infected with the recovered virus stocks from either pLC16m8.OS-BAC (vLC16m8.OS-BAC) or pLC16m8.8S-BAC (vLC16m8.8S-BAC). The plaque size and EGFP expression of vLC16m8.OS-BAC (plaque A and B) and vLC16m8.8S-BAC (plaque C and D) were observed at the same magnification under phase-contrast with fluorescent microscopy at 2 d.p.i.

    Journal: PLoS ONE

    Article Title: Construction and characterization of bacterial artificial chromosomes harboring the full-length genome of a highly attenuated vaccinia virus LC16m8

    doi: 10.1371/journal.pone.0192725

    Figure Lengend Snippet: The characteristics of the viruses recovered from the pLC16m8.8S-BAC or pLC16m8.OS-BAC. The plaque size and EGFP expression were observed under phase-contrast and fluorescent microscopy. RK13 cells were infected with the recovered virus stocks from either pLC16m8.OS-BAC (vLC16m8.OS-BAC) or pLC16m8.8S-BAC (vLC16m8.8S-BAC). The plaque size and EGFP expression of vLC16m8.OS-BAC (plaque A and B) and vLC16m8.8S-BAC (plaque C and D) were observed at the same magnification under phase-contrast with fluorescent microscopy at 2 d.p.i.

    Article Snippet: First, 8.5 μl of m8-EGFP-BAC stock solution (approximately 106 to 107 pfu/ml of viruses in DMEM-5 FBS) was mixed with 1 μl of 10 x Proteinase K lysis buffer (100 mM Tris-Cl; pH 8.0, 4.5% NP40, and 4.5% Tween 20) and 0.5 μl of 20 mg/ml proteinase K (Takara, Shiga, Japan).

    Techniques: BAC Assay, Expressing, Microscopy, Infection