dntp  (Thermo Fisher)


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    Name:
    dNTP Mix
    Description:
    This dNTP mix consists of four nucleotides dATP dCTP dGTP dTTP 2 deoxynucleoside 5 triphosphates each at a concentration of 10 mM in a solution of 0 6 mM Tris HCl pH 7 5 This mix is suitable for use in PCR sequencing fill in nick translation cDNA synthesis TdT tailing reactions and dilution of radiolabeled dNTPs
    Catalog Number:
    18427013
    Price:
    None
    Category:
    Oligos Primers Probes Nucleotides
    Applications:
    PCR|PCR & Real-Time PCR|PCR Buffers, dNTPs, MgCl2 & General Reagents
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    Structured Review

    Thermo Fisher dntp
    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM <t>MgCl2,</t> 50 mM KCl, 0.1% Triton X-100, 200 μM each <t>dNTP,</t> 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
    This dNTP mix consists of four nucleotides dATP dCTP dGTP dTTP 2 deoxynucleoside 5 triphosphates each at a concentration of 10 mM in a solution of 0 6 mM Tris HCl pH 7 5 This mix is suitable for use in PCR sequencing fill in nick translation cDNA synthesis TdT tailing reactions and dilution of radiolabeled dNTPs
    https://www.bioz.com/result/dntp/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dntp - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism"

    Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-4-21

    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
    Figure Legend Snippet: Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Size-exclusion Chromatography, Agarose Gel Electrophoresis, Purification, Incubation, Ligation, Clone Assay

    Related Articles

    Transduction:

    Article Title: Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs
    Article Snippet: SAMHD1 shRNA, encoded within a SIN vector cassette (St. Louis, MO, TRCN0000145408), and control mammalian non-target shRNA (SHC002) were purchased from Sigma-Aldrich. .. THP-1 cells transduced with lentiviral vectors encoding SAMHD1 shRNA were selected by culturing in a medium containing 0.8 μg/ml puromycin. dNs consisted of a mixture of dA (D8668), dC (D0776), dG (D0901) and dT (T1895; all from Sigma-Aldrich). dNTPs (R0192) were purchased from Fermentas (Glen Burnie, MD). .. Cytokine cocktail for HSPC transduction consisted of a mixture of stem cell factor (SCF) at 100 ng/ml, Fms-related tyrosine kinase 3 ligand (Flt3L) at 100 ng/ml, interleukin-3 (IL-3) at 60 ng/ml and thrombopoietin (TPO) at 10 ng/ml (Immunotools, Friesoythe, Germany).

    shRNA:

    Article Title: Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs
    Article Snippet: SAMHD1 shRNA, encoded within a SIN vector cassette (St. Louis, MO, TRCN0000145408), and control mammalian non-target shRNA (SHC002) were purchased from Sigma-Aldrich. .. THP-1 cells transduced with lentiviral vectors encoding SAMHD1 shRNA were selected by culturing in a medium containing 0.8 μg/ml puromycin. dNs consisted of a mixture of dA (D8668), dC (D0776), dG (D0901) and dT (T1895; all from Sigma-Aldrich). dNTPs (R0192) were purchased from Fermentas (Glen Burnie, MD). .. Cytokine cocktail for HSPC transduction consisted of a mixture of stem cell factor (SCF) at 100 ng/ml, Fms-related tyrosine kinase 3 ligand (Flt3L) at 100 ng/ml, interleukin-3 (IL-3) at 60 ng/ml and thrombopoietin (TPO) at 10 ng/ml (Immunotools, Friesoythe, Germany).

    Polymerase Chain Reaction:

    Article Title: A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering
    Article Snippet: The reaction was performed with a final concentration of 0.4 mM dATP, 0.4 mM dTTP, 0.4 mM dCTP, 0.4 mM dGTP, 20 mM Tris/HCl pH 8.8, 10 mM KCl, 6 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1 mg/ml BSA and 0.1% Triton X-100. .. PCR with dUTP replacing dTTP was performed using standard Phusion (Finnzymes) reaction conditions as recommended by the manufacturer, using the dUTP/dNTPs mix (Fermentas) instead of dNTPs. .. The pET-Pfu-vector was a kind gift from Jens Lykke-Andersen.

    Article Title: Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq
    Article Snippet: Formaldehyde (sigma, Cat#: F8775) Mineral Oil (Sigma, Cat. #: M5904) Proteinase K (Invitrogen, Cat#. .. 25530-031) Protease inhibitor cocktail (Roche, Cat.#: 11836153001): Phenol:Chloroform:Isoamyl Alchohol (Invitrogen, Cat. #:15593031) RNAse (Invitrogen, Cat. #:12091-021) Sequenase Enzyme Kit (Version 2.0, US Biochemical, Cat. #: 70775 dNTP mix (Invitrogen, Cat.#: 18427-013 ) BSA (10 mg/ml , NEB, #B9001S) DTT (0.1 M stock, Invitrogen, #:Y00147) Primer 1 (4 μM): 5'-GACATGTATCCGGATGTNNNNNNNNN-3' ExoSAP-IT (USB, Cat.#:78200) Phusion Polymerase (NEB, Cat#: F-530L, provided with 100 % DMSO, 5X GC buffer) Primer 2 (10 μM stock): 5'-GACATGTATCCGGATGT-3’ BciVI Restriction Enzyme (NEB, Cat. #: R0596L) Agarose (Fisher, Cat #: BP160-500) Protein A-sepharose beads (Sigma, Cat#: P9424) PCR purification, Gel extraction and reaction clean up kits (Qiagen, Cat#s: 28006, 28606, 28206) Glycogen (20μg/μl, Invitrogen, Cat#: 10814-010) Quant-It dsDNA HS assay kit (Invitrogen, Cat#: ) .. 0.2 ml PCR tube strips (Eppendorf, Cat. #: 951010022) 1.5 ml tubes low retention tubes (Eppendorf, Cat. #:022431021) 15 ml tubes (BD Falcon cat. no. 352097) Filtered micropipette tips (Danville Sci., Cat.#s:P1096FR, P1121, P1122, P1126 ) Micropipiettes (1-1000 μl range) Spin-X wash columns (Costar, Cat#:8160) Refrigerated microcentrifuge (Eppendorf model 5415R or equivalent) Minicentrifuge with strip tubes adapter (Fisher Scientific Cat. #:05090-100) Cold room Sonicator (Branson 250 Digitial sonifier) Rotating mixer for 1.5 ml tubes Vortex mixer Thermal Cycler (Eppendorf, master cycle gradient or equivalent) Agarose gel electrophoresis equipment Qubit Flourometer (Invitrogen, Cat #: 32857)

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: ThermoPol Reaction Buffer (New England Biolabs, Cat. # M0254S) Wash Buffer: 10 mM Tris–HCl (pH 8.2), 1 M NaCl, 1 mM EDTA Binding Buffer: 10 mM Tris–HCl (pH 8.2), 1 M NaCl, 1 mM EDTA, 0.2% Tween20 Elution Buffer: 30 mM Tris–HCl (pH 7), 100 mM KCl, 1 mM EDTA .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

    Article Title: rpoB Gene Analysis as a Novel Strategy for Identification of Spirochetes from the Genera Borrelia, Treponema, and Leptospira
    Article Snippet: Oligonucleotide primers were synthesized in our laboratory (392 DNA/RNA Synthesizer; Perkin-Elmer). .. Amplification of rpoB fragments was carried out by using the LTB primers listed in Table (3.2 pmol each) added to a 50-μl PCR mix containing 10 mM dNTP, 1 U of Taq polymerase (Gibco BRL), and 2 μl of DNA extract. ..

    Article Title: Analysis of Endocannabinoid System in Rat Testis During the First Spermatogenetic Wave
    Article Snippet: As a negative control, total RNA (2 µg) was incubated in the previous mix without adding Reverse Transcriptase enzyme (RT− cDNA sample). .. PCR was carried out using 2 µl cDNA and 10 pmol of the appropriate primers in a PCR mix [0.2 mM dNTP, 1× PCR buffer (Invitrogen Life Technologies), 1.5 mM MgCl2 , 1.25 U Taq Polymerase (Invitrogen Life Technologies, Paisley, UK)], using an Applied Biosystem Thermocycler. .. Possible residual genomic DNA was evaluated using Actin primers by RT− cDNA amplification (at 35 circles) while possible contamination among samples was excluded using samples prepared with 1 µl water (H2 O samples, negative control) in place of cDNA.

    Article Title: Interleukin-17A is involved in mechanical hyperalgesia but not in the severity of murine antigen-induced arthritis
    Article Snippet: For PCR (MyCycler Thermal Cycler, Bio-Rad, Hercules, CA, USA) we used Phire Hot Start II DNA Polymerase (Thermo Fisher Scientific). .. The Master Mix consisted of 1x PCR buffer, 1 unit Taq DNA polymerase per 50 µl PCR mixture, 0.2 mM dNTP mixture (10 m M dNTP Mix; Thermo Fisher Scientific), and 0.5 µM primer. .. For amplification, 20–24 µl of Master Mix plus 1–5 µl cDNA template were used.

    Protease Inhibitor:

    Article Title: Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq
    Article Snippet: Formaldehyde (sigma, Cat#: F8775) Mineral Oil (Sigma, Cat. #: M5904) Proteinase K (Invitrogen, Cat#. .. 25530-031) Protease inhibitor cocktail (Roche, Cat.#: 11836153001): Phenol:Chloroform:Isoamyl Alchohol (Invitrogen, Cat. #:15593031) RNAse (Invitrogen, Cat. #:12091-021) Sequenase Enzyme Kit (Version 2.0, US Biochemical, Cat. #: 70775 dNTP mix (Invitrogen, Cat.#: 18427-013 ) BSA (10 mg/ml , NEB, #B9001S) DTT (0.1 M stock, Invitrogen, #:Y00147) Primer 1 (4 μM): 5'-GACATGTATCCGGATGTNNNNNNNNN-3' ExoSAP-IT (USB, Cat.#:78200) Phusion Polymerase (NEB, Cat#: F-530L, provided with 100 % DMSO, 5X GC buffer) Primer 2 (10 μM stock): 5'-GACATGTATCCGGATGT-3’ BciVI Restriction Enzyme (NEB, Cat. #: R0596L) Agarose (Fisher, Cat #: BP160-500) Protein A-sepharose beads (Sigma, Cat#: P9424) PCR purification, Gel extraction and reaction clean up kits (Qiagen, Cat#s: 28006, 28606, 28206) Glycogen (20μg/μl, Invitrogen, Cat#: 10814-010) Quant-It dsDNA HS assay kit (Invitrogen, Cat#: ) .. 0.2 ml PCR tube strips (Eppendorf, Cat. #: 951010022) 1.5 ml tubes low retention tubes (Eppendorf, Cat. #:022431021) 15 ml tubes (BD Falcon cat. no. 352097) Filtered micropipette tips (Danville Sci., Cat.#s:P1096FR, P1121, P1122, P1126 ) Micropipiettes (1-1000 μl range) Spin-X wash columns (Costar, Cat#:8160) Refrigerated microcentrifuge (Eppendorf model 5415R or equivalent) Minicentrifuge with strip tubes adapter (Fisher Scientific Cat. #:05090-100) Cold room Sonicator (Branson 250 Digitial sonifier) Rotating mixer for 1.5 ml tubes Vortex mixer Thermal Cycler (Eppendorf, master cycle gradient or equivalent) Agarose gel electrophoresis equipment Qubit Flourometer (Invitrogen, Cat #: 32857)

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq
    Article Snippet: Formaldehyde (sigma, Cat#: F8775) Mineral Oil (Sigma, Cat. #: M5904) Proteinase K (Invitrogen, Cat#. .. 25530-031) Protease inhibitor cocktail (Roche, Cat.#: 11836153001): Phenol:Chloroform:Isoamyl Alchohol (Invitrogen, Cat. #:15593031) RNAse (Invitrogen, Cat. #:12091-021) Sequenase Enzyme Kit (Version 2.0, US Biochemical, Cat. #: 70775 dNTP mix (Invitrogen, Cat.#: 18427-013 ) BSA (10 mg/ml , NEB, #B9001S) DTT (0.1 M stock, Invitrogen, #:Y00147) Primer 1 (4 μM): 5'-GACATGTATCCGGATGTNNNNNNNNN-3' ExoSAP-IT (USB, Cat.#:78200) Phusion Polymerase (NEB, Cat#: F-530L, provided with 100 % DMSO, 5X GC buffer) Primer 2 (10 μM stock): 5'-GACATGTATCCGGATGT-3’ BciVI Restriction Enzyme (NEB, Cat. #: R0596L) Agarose (Fisher, Cat #: BP160-500) Protein A-sepharose beads (Sigma, Cat#: P9424) PCR purification, Gel extraction and reaction clean up kits (Qiagen, Cat#s: 28006, 28606, 28206) Glycogen (20μg/μl, Invitrogen, Cat#: 10814-010) Quant-It dsDNA HS assay kit (Invitrogen, Cat#: ) .. 0.2 ml PCR tube strips (Eppendorf, Cat. #: 951010022) 1.5 ml tubes low retention tubes (Eppendorf, Cat. #:022431021) 15 ml tubes (BD Falcon cat. no. 352097) Filtered micropipette tips (Danville Sci., Cat.#s:P1096FR, P1121, P1122, P1126 ) Micropipiettes (1-1000 μl range) Spin-X wash columns (Costar, Cat#:8160) Refrigerated microcentrifuge (Eppendorf model 5415R or equivalent) Minicentrifuge with strip tubes adapter (Fisher Scientific Cat. #:05090-100) Cold room Sonicator (Branson 250 Digitial sonifier) Rotating mixer for 1.5 ml tubes Vortex mixer Thermal Cycler (Eppendorf, master cycle gradient or equivalent) Agarose gel electrophoresis equipment Qubit Flourometer (Invitrogen, Cat #: 32857)

    Purification:

    Article Title: Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq
    Article Snippet: Formaldehyde (sigma, Cat#: F8775) Mineral Oil (Sigma, Cat. #: M5904) Proteinase K (Invitrogen, Cat#. .. 25530-031) Protease inhibitor cocktail (Roche, Cat.#: 11836153001): Phenol:Chloroform:Isoamyl Alchohol (Invitrogen, Cat. #:15593031) RNAse (Invitrogen, Cat. #:12091-021) Sequenase Enzyme Kit (Version 2.0, US Biochemical, Cat. #: 70775 dNTP mix (Invitrogen, Cat.#: 18427-013 ) BSA (10 mg/ml , NEB, #B9001S) DTT (0.1 M stock, Invitrogen, #:Y00147) Primer 1 (4 μM): 5'-GACATGTATCCGGATGTNNNNNNNNN-3' ExoSAP-IT (USB, Cat.#:78200) Phusion Polymerase (NEB, Cat#: F-530L, provided with 100 % DMSO, 5X GC buffer) Primer 2 (10 μM stock): 5'-GACATGTATCCGGATGT-3’ BciVI Restriction Enzyme (NEB, Cat. #: R0596L) Agarose (Fisher, Cat #: BP160-500) Protein A-sepharose beads (Sigma, Cat#: P9424) PCR purification, Gel extraction and reaction clean up kits (Qiagen, Cat#s: 28006, 28606, 28206) Glycogen (20μg/μl, Invitrogen, Cat#: 10814-010) Quant-It dsDNA HS assay kit (Invitrogen, Cat#: ) .. 0.2 ml PCR tube strips (Eppendorf, Cat. #: 951010022) 1.5 ml tubes low retention tubes (Eppendorf, Cat. #:022431021) 15 ml tubes (BD Falcon cat. no. 352097) Filtered micropipette tips (Danville Sci., Cat.#s:P1096FR, P1121, P1122, P1126 ) Micropipiettes (1-1000 μl range) Spin-X wash columns (Costar, Cat#:8160) Refrigerated microcentrifuge (Eppendorf model 5415R or equivalent) Minicentrifuge with strip tubes adapter (Fisher Scientific Cat. #:05090-100) Cold room Sonicator (Branson 250 Digitial sonifier) Rotating mixer for 1.5 ml tubes Vortex mixer Thermal Cycler (Eppendorf, master cycle gradient or equivalent) Agarose gel electrophoresis equipment Qubit Flourometer (Invitrogen, Cat #: 32857)

    Gel Extraction:

    Article Title: Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq
    Article Snippet: Formaldehyde (sigma, Cat#: F8775) Mineral Oil (Sigma, Cat. #: M5904) Proteinase K (Invitrogen, Cat#. .. 25530-031) Protease inhibitor cocktail (Roche, Cat.#: 11836153001): Phenol:Chloroform:Isoamyl Alchohol (Invitrogen, Cat. #:15593031) RNAse (Invitrogen, Cat. #:12091-021) Sequenase Enzyme Kit (Version 2.0, US Biochemical, Cat. #: 70775 dNTP mix (Invitrogen, Cat.#: 18427-013 ) BSA (10 mg/ml , NEB, #B9001S) DTT (0.1 M stock, Invitrogen, #:Y00147) Primer 1 (4 μM): 5'-GACATGTATCCGGATGTNNNNNNNNN-3' ExoSAP-IT (USB, Cat.#:78200) Phusion Polymerase (NEB, Cat#: F-530L, provided with 100 % DMSO, 5X GC buffer) Primer 2 (10 μM stock): 5'-GACATGTATCCGGATGT-3’ BciVI Restriction Enzyme (NEB, Cat. #: R0596L) Agarose (Fisher, Cat #: BP160-500) Protein A-sepharose beads (Sigma, Cat#: P9424) PCR purification, Gel extraction and reaction clean up kits (Qiagen, Cat#s: 28006, 28606, 28206) Glycogen (20μg/μl, Invitrogen, Cat#: 10814-010) Quant-It dsDNA HS assay kit (Invitrogen, Cat#: ) .. 0.2 ml PCR tube strips (Eppendorf, Cat. #: 951010022) 1.5 ml tubes low retention tubes (Eppendorf, Cat. #:022431021) 15 ml tubes (BD Falcon cat. no. 352097) Filtered micropipette tips (Danville Sci., Cat.#s:P1096FR, P1121, P1122, P1126 ) Micropipiettes (1-1000 μl range) Spin-X wash columns (Costar, Cat#:8160) Refrigerated microcentrifuge (Eppendorf model 5415R or equivalent) Minicentrifuge with strip tubes adapter (Fisher Scientific Cat. #:05090-100) Cold room Sonicator (Branson 250 Digitial sonifier) Rotating mixer for 1.5 ml tubes Vortex mixer Thermal Cycler (Eppendorf, master cycle gradient or equivalent) Agarose gel electrophoresis equipment Qubit Flourometer (Invitrogen, Cat #: 32857)

    Plasmid Preparation:

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: ThermoPol Reaction Buffer (New England Biolabs, Cat. # M0254S) Wash Buffer: 10 mM Tris–HCl (pH 8.2), 1 M NaCl, 1 mM EDTA Binding Buffer: 10 mM Tris–HCl (pH 8.2), 1 M NaCl, 1 mM EDTA, 0.2% Tween20 Elution Buffer: 30 mM Tris–HCl (pH 7), 100 mM KCl, 1 mM EDTA .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

    Amplification:

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: ThermoPol Reaction Buffer (New England Biolabs, Cat. # M0254S) Wash Buffer: 10 mM Tris–HCl (pH 8.2), 1 M NaCl, 1 mM EDTA Binding Buffer: 10 mM Tris–HCl (pH 8.2), 1 M NaCl, 1 mM EDTA, 0.2% Tween20 Elution Buffer: 30 mM Tris–HCl (pH 7), 100 mM KCl, 1 mM EDTA .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

    Article Title: rpoB Gene Analysis as a Novel Strategy for Identification of Spirochetes from the Genera Borrelia, Treponema, and Leptospira
    Article Snippet: Oligonucleotide primers were synthesized in our laboratory (392 DNA/RNA Synthesizer; Perkin-Elmer). .. Amplification of rpoB fragments was carried out by using the LTB primers listed in Table (3.2 pmol each) added to a 50-μl PCR mix containing 10 mM dNTP, 1 U of Taq polymerase (Gibco BRL), and 2 μl of DNA extract. ..

    Synthesized:

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: ThermoPol Reaction Buffer (New England Biolabs, Cat. # M0254S) Wash Buffer: 10 mM Tris–HCl (pH 8.2), 1 M NaCl, 1 mM EDTA Binding Buffer: 10 mM Tris–HCl (pH 8.2), 1 M NaCl, 1 mM EDTA, 0.2% Tween20 Elution Buffer: 30 mM Tris–HCl (pH 7), 100 mM KCl, 1 mM EDTA .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

    Staining:

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: ThermoPol Reaction Buffer (New England Biolabs, Cat. # M0254S) Wash Buffer: 10 mM Tris–HCl (pH 8.2), 1 M NaCl, 1 mM EDTA Binding Buffer: 10 mM Tris–HCl (pH 8.2), 1 M NaCl, 1 mM EDTA, 0.2% Tween20 Elution Buffer: 30 mM Tris–HCl (pH 7), 100 mM KCl, 1 mM EDTA .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

    Lysis:

    Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling
    Article Snippet: Cell lysis Cells were sorted with a BD FACSAria (Becton Dickinson) into 96-well plates (Applied Biosystems) with 5 μl lysis buffer per well as described ( , ). .. The following chemicals were evaluated (final lysis concentrations are shown): 7-deaza-2′-deoxyguanosine-5′-triphosphate lithium salt (100 μM, Sigma-Aldrich); Betaine solution (4 M, Sigma-Aldrich); BSA (1–4 mg/ml, Fermentas); guanidine thiocyanate solution (GTC) (40–80 mM, Sigma-Aldrich); GenElute linear polyacrylamide (LPA) (50 ng/μl, Sigma-Aldrich); Igepal CA-630 (also known as Non-idet P-40, 0.5–4%, Sigma-Aldrich); polyinosinic acid potassium salt (50 ng/μl, Sigma-Aldrich); RNAseOUT (10 U/μl, Invitrogen); 2× reverse transcription buffer: 100 mM Tris-HCl (pH 8.3), 150 mM KCl, and 6 mM MgCl2 (Invitrogen); d -(+)-trehalose dihydrate (1 M, Sigma-Aldrich); yeast tRNA (50 ng/μl, Ambion); RT mix (2× RT buffer, Invitrogen, 5 μM random hexamers (Metabion), 5 μM oligo-dT (Metabion), 1 mM dNTP); RT mix with BSA (2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, 1 mM dNTP, 1 mg/ml BSA); and RNase-free water (Gibco). ..

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  • 99
    Thermo Fisher complementary dna oligonucleotides
    PCR-based analysis of SPRY1 gene mutations. (a) PCR-based mutation detection assay according to the protocol provided by the Guide-it Mutation Detection Kit from Takara Bio Inc . (USA): The genomic region covering the expected <t>CRISPR/Cas9-mediated</t> mutations within the SPRY1 gene was PCR-amplified (see, Figure 1(b )) in CRISPR/Cas9-GFP (GFP), CRISPR/Cas9-SPRY1 #1 (#1) and CRISPR/Cas9-SPRY1 #2 (#2) expressing ASCs. The PCR product (972bp) obtained from each cell population was melted and re-hybridized. This step results in the formation of <t>DNA</t> heteroduplexes originating from wildtype and mutated cells. Subsequently, mismatched DNA strands were cleaved by Resolvase and analysed by agarose gel electrophoreses. Predicted cleavage products are indicated by arrows. CRISPR/Cas9 efficiency was calculated as described in the methods section. N. d.: not determined; (b) Tracking of indels by decomposition (TIDE). The PCR product obtained in a was purified and Sanger sequenced using the forward primer. Distribution of indels within the targeted SPRY1 gene locus was calculated using the TIDE algorithm as described in the methods section. A representative result of n = 4 donors is shown
    Complementary Dna Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher dntps
    Overview of Luminex procedure for determining plasmid content. (1) Plasmid-specific regions are amplified by multiplex PCR, using genomic DNA or B. burgdorferi culture as the template. Unincorporated primers and <t>dNTPs</t> in amplified PCR products are removed by treatment with exonuclease I and alkaline phosphatase. (2) Primers that contain an xTAG sequence are utilized in an asymmetric PCR that incorporates <t>biotin-dCTP.</t> (3) Biotinylated products are hybridized with xTAG microspheres that are coupled to antitag sequences and detected by binding of streptavidin-R-phycoerythrin. MFI, mean fluorescence intensity.
    Dntps, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dntp
    Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM <t>oligo-dT,</t> and 1 mM <t>dNTP.</t>
    Dntp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PCR-based analysis of SPRY1 gene mutations. (a) PCR-based mutation detection assay according to the protocol provided by the Guide-it Mutation Detection Kit from Takara Bio Inc . (USA): The genomic region covering the expected CRISPR/Cas9-mediated mutations within the SPRY1 gene was PCR-amplified (see, Figure 1(b )) in CRISPR/Cas9-GFP (GFP), CRISPR/Cas9-SPRY1 #1 (#1) and CRISPR/Cas9-SPRY1 #2 (#2) expressing ASCs. The PCR product (972bp) obtained from each cell population was melted and re-hybridized. This step results in the formation of DNA heteroduplexes originating from wildtype and mutated cells. Subsequently, mismatched DNA strands were cleaved by Resolvase and analysed by agarose gel electrophoreses. Predicted cleavage products are indicated by arrows. CRISPR/Cas9 efficiency was calculated as described in the methods section. N. d.: not determined; (b) Tracking of indels by decomposition (TIDE). The PCR product obtained in a was purified and Sanger sequenced using the forward primer. Distribution of indels within the targeted SPRY1 gene locus was calculated using the TIDE algorithm as described in the methods section. A representative result of n = 4 donors is shown

    Journal: Adipocyte

    Article Title: CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells

    doi: 10.1080/21623945.2020.1834230

    Figure Lengend Snippet: PCR-based analysis of SPRY1 gene mutations. (a) PCR-based mutation detection assay according to the protocol provided by the Guide-it Mutation Detection Kit from Takara Bio Inc . (USA): The genomic region covering the expected CRISPR/Cas9-mediated mutations within the SPRY1 gene was PCR-amplified (see, Figure 1(b )) in CRISPR/Cas9-GFP (GFP), CRISPR/Cas9-SPRY1 #1 (#1) and CRISPR/Cas9-SPRY1 #2 (#2) expressing ASCs. The PCR product (972bp) obtained from each cell population was melted and re-hybridized. This step results in the formation of DNA heteroduplexes originating from wildtype and mutated cells. Subsequently, mismatched DNA strands were cleaved by Resolvase and analysed by agarose gel electrophoreses. Predicted cleavage products are indicated by arrows. CRISPR/Cas9 efficiency was calculated as described in the methods section. N. d.: not determined; (b) Tracking of indels by decomposition (TIDE). The PCR product obtained in a was purified and Sanger sequenced using the forward primer. Distribution of indels within the targeted SPRY1 gene locus was calculated using the TIDE algorithm as described in the methods section. A representative result of n = 4 donors is shown

    Article Snippet: Complementary DNA oligonucleotides containing selected CRISPR/Cas9 target sequences (Supplementary Table 3) were phosphorylated, annealed and ligated into the vector.

    Techniques: Polymerase Chain Reaction, Mutagenesis, Detection Assay, CRISPR, Amplification, Expressing, Agarose Gel Electrophoresis, Purification

    Overview of Luminex procedure for determining plasmid content. (1) Plasmid-specific regions are amplified by multiplex PCR, using genomic DNA or B. burgdorferi culture as the template. Unincorporated primers and dNTPs in amplified PCR products are removed by treatment with exonuclease I and alkaline phosphatase. (2) Primers that contain an xTAG sequence are utilized in an asymmetric PCR that incorporates biotin-dCTP. (3) Biotinylated products are hybridized with xTAG microspheres that are coupled to antitag sequences and detected by binding of streptavidin-R-phycoerythrin. MFI, mean fluorescence intensity.

    Journal: Applied and Environmental Microbiology

    Article Title: High-Throughput Plasmid Content Analysis of Borrelia burgdorferi B31 by Using Luminex Multiplex Technology ▿ B31 by Using Luminex Multiplex Technology ▿ †

    doi: 10.1128/AEM.01877-10

    Figure Lengend Snippet: Overview of Luminex procedure for determining plasmid content. (1) Plasmid-specific regions are amplified by multiplex PCR, using genomic DNA or B. burgdorferi culture as the template. Unincorporated primers and dNTPs in amplified PCR products are removed by treatment with exonuclease I and alkaline phosphatase. (2) Primers that contain an xTAG sequence are utilized in an asymmetric PCR that incorporates biotin-dCTP. (3) Biotinylated products are hybridized with xTAG microspheres that are coupled to antitag sequences and detected by binding of streptavidin-R-phycoerythrin. MFI, mean fluorescence intensity.

    Article Snippet: The ASPE reaction mixture consisted of 10.1 μl of nuclease-free water, 2 μl of 10× PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl), 1 μl of pooled tag-ASPE primers (8 primers per pool; 500 nM [each]), 1 μl of 20× dNTPs (without dCTP; 1 μM [each]), 0.5 μl 50 mM MgCl2 , 0.25 μl 0.4 mM biotin-dCTP (Invitrogen), 0.15 μl of Platinum GenoTYPE Tsp DNA polymerase (Invitrogen), and 5 μl of ExoSapIT-treated PCR product.

    Techniques: Luminex, Plasmid Preparation, Amplification, Multiplex Assay, Polymerase Chain Reaction, Sequencing, Binding Assay, Fluorescence

    Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Journal: Frontiers in Oncology

    Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling

    doi: 10.3389/fonc.2013.00274

    Figure Lengend Snippet: Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Article Snippet: The following chemicals were evaluated (final lysis concentrations are shown): 7-deaza-2′-deoxyguanosine-5′-triphosphate lithium salt (100 μM, Sigma-Aldrich); Betaine solution (4 M, Sigma-Aldrich); BSA (1–4 mg/ml, Fermentas); guanidine thiocyanate solution (GTC) (40–80 mM, Sigma-Aldrich); GenElute linear polyacrylamide (LPA) (50 ng/μl, Sigma-Aldrich); Igepal CA-630 (also known as Non-idet P-40, 0.5–4%, Sigma-Aldrich); polyinosinic acid potassium salt (50 ng/μl, Sigma-Aldrich); RNAseOUT (10 U/μl, Invitrogen); 2× reverse transcription buffer: 100 mM Tris-HCl (pH 8.3), 150 mM KCl, and 6 mM MgCl2 (Invitrogen); d -(+)-trehalose dihydrate (1 M, Sigma-Aldrich); yeast tRNA (50 ng/μl, Ambion); RT mix (2× RT buffer, Invitrogen, 5 μM random hexamers (Metabion), 5 μM oligo-dT (Metabion), 1 mM dNTP); RT mix with BSA (2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, 1 mM dNTP, 1 mg/ml BSA); and RNase-free water (Gibco).

    Techniques: Lysis, Quantitative RT-PCR, Purification, Polymerase Chain Reaction

    Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Journal: Frontiers in Oncology

    Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling

    doi: 10.3389/fonc.2013.00274

    Figure Lengend Snippet: Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Article Snippet: The following chemicals were evaluated (final lysis concentrations are shown): 7-deaza-2′-deoxyguanosine-5′-triphosphate lithium salt (100 μM, Sigma-Aldrich); Betaine solution (4 M, Sigma-Aldrich); BSA (1–4 mg/ml, Fermentas); guanidine thiocyanate solution (GTC) (40–80 mM, Sigma-Aldrich); GenElute linear polyacrylamide (LPA) (50 ng/μl, Sigma-Aldrich); Igepal CA-630 (also known as Non-idet P-40, 0.5–4%, Sigma-Aldrich); polyinosinic acid potassium salt (50 ng/μl, Sigma-Aldrich); RNAseOUT (10 U/μl, Invitrogen); 2× reverse transcription buffer: 100 mM Tris-HCl (pH 8.3), 150 mM KCl, and 6 mM MgCl2 (Invitrogen); d -(+)-trehalose dihydrate (1 M, Sigma-Aldrich); yeast tRNA (50 ng/μl, Ambion); RT mix (2× RT buffer, Invitrogen, 5 μM random hexamers (Metabion), 5 μM oligo-dT (Metabion), 1 mM dNTP); RT mix with BSA (2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, 1 mM dNTP, 1 mg/ml BSA); and RNase-free water (Gibco).

    Techniques: Lysis, Polymerase Chain Reaction

    Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.

    Journal: Methods in cell biology

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo

    doi: 10.1016/bs.mcb.2015.01.021

    Figure Lengend Snippet: Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.

    Article Snippet: pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol.

    Techniques: Labeling, Plasmid Preparation, Flow Cytometry, Ligation