amv reverse transcriptase  (New England Biolabs)


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    Name:
    AMV Reverse Transcriptase
    Description:
    AMV Reverse Transcriptase 1 000 units
    Catalog Number:
    m0277l
    Price:
    292
    Size:
    1 000 units
    Category:
    Reverse Transcriptases
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    Structured Review

    New England Biolabs amv reverse transcriptase
    AMV Reverse Transcriptase
    AMV Reverse Transcriptase 1 000 units
    https://www.bioz.com/result/amv reverse transcriptase/product/New England Biolabs
    Average 95 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    amv reverse transcriptase - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes"

    Article Title: Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw028

    Reverse transcription using AHP dUTP. ( A ) RNA template T5 with primer P3. ( B and C ) Twenty percent denaturing PAGE analysis of reactions using AMV and M-MuLV (RNase H-) reverse transcriptases at 42°C for 15 h.
    Figure Legend Snippet: Reverse transcription using AHP dUTP. ( A ) RNA template T5 with primer P3. ( B and C ) Twenty percent denaturing PAGE analysis of reactions using AMV and M-MuLV (RNase H-) reverse transcriptases at 42°C for 15 h.

    Techniques Used: Polyacrylamide Gel Electrophoresis

    2) Product Images from "Two Tandem RNase III Cleavage Sites Determine betT mRNA Stability in Response to Osmotic Stress in Escherichia coli"

    Article Title: Two Tandem RNase III Cleavage Sites Determine betT mRNA Stability in Response to Osmotic Stress in Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0100520

    Identification of RNase III cleavage sites in betT mRNA in vitro and in vivo . (A) In vitro cleavage of the full-length synthetic betT mRNA. The 5′-end-labeled betT transcript (4 pmol) was incubated with purified RNase III (1 pmol) in cleavage buffer with (+) or without (-) MgCl 2 at 37°C. The size of the cleavage products was estimated using size markers generated by internally labeled transcripts. The major cleavage products are indicated with arrows. Other minor cleavage products are indicated with asterisks. (B) Primer extension analysis of betT mRNA. Total RNA was prepared from MG1655 rnc harboring pBetRS1 and either pKAN6B or pRNC1, which exogenously overexpressed betT mRNA, hybridized with a 5′- 32 P-end-labeled primer (betT+120R), and extended using AMV reverse transcriptase. Sequencing ladders were produced using the same primer used in cDNA synthesis and PCR DNA, encompassing the betT gene as a template. (C) The predicted secondary structure of betT mRNA region encompassing RNase III cleavage sites. The secondary structure was determined using the M-fold program [33] . (D) In vitro cleavage of the model hairpin RNA of betT mRNA. 3′-end-labeled betT model hairpin (25 pmol) was incubated with purified RNase III (1 pmol) in a cleavage buffer with (+) or without (−) MgCl 2 , respectively. Cleavage products (I, II, III, and IV) were identified using size markers generated by alkaline hydrolysis and RNase T1 digestion. Relative abundance of each cleavage product was assessed by measuring the radioactivity of each band and plotted.
    Figure Legend Snippet: Identification of RNase III cleavage sites in betT mRNA in vitro and in vivo . (A) In vitro cleavage of the full-length synthetic betT mRNA. The 5′-end-labeled betT transcript (4 pmol) was incubated with purified RNase III (1 pmol) in cleavage buffer with (+) or without (-) MgCl 2 at 37°C. The size of the cleavage products was estimated using size markers generated by internally labeled transcripts. The major cleavage products are indicated with arrows. Other minor cleavage products are indicated with asterisks. (B) Primer extension analysis of betT mRNA. Total RNA was prepared from MG1655 rnc harboring pBetRS1 and either pKAN6B or pRNC1, which exogenously overexpressed betT mRNA, hybridized with a 5′- 32 P-end-labeled primer (betT+120R), and extended using AMV reverse transcriptase. Sequencing ladders were produced using the same primer used in cDNA synthesis and PCR DNA, encompassing the betT gene as a template. (C) The predicted secondary structure of betT mRNA region encompassing RNase III cleavage sites. The secondary structure was determined using the M-fold program [33] . (D) In vitro cleavage of the model hairpin RNA of betT mRNA. 3′-end-labeled betT model hairpin (25 pmol) was incubated with purified RNase III (1 pmol) in a cleavage buffer with (+) or without (−) MgCl 2 , respectively. Cleavage products (I, II, III, and IV) were identified using size markers generated by alkaline hydrolysis and RNase T1 digestion. Relative abundance of each cleavage product was assessed by measuring the radioactivity of each band and plotted.

    Techniques Used: In Vitro, In Vivo, Labeling, Incubation, Purification, Generated, Sequencing, Produced, Polymerase Chain Reaction, Radioactivity

    Related Articles

    Clone Assay:

    Article Title: An in vivo selection method to optimize trans-splicing ribozymes
    Article Snippet: Three separate clones were used for each ribozyme. .. The transcriptions were incubated for 1 h at 42°C with AMV reverse transcriptase (NEB).

    Article Title: Simian Rotaviruses Possess Divergent Gene Constellations That Originated from Interspecies Transmission and Reassortment ▿
    Article Snippet: Afterward, the dsRNA segments were resolved by electrophoresis on a 1% agarose gel, and each was recovered by using a QIAquick gel extraction kit (Qiagen). cDNAs were synthesized for each dsRNA segment using AMV reverse transcriptase (New England Biolabs) and then PCR amplified with ExTaq DNA polymerase (Takara). .. The cDNAs were cloned into the pGEM-T Easy vector (Promega).

    Amplification:

    Article Title: Programmed genome rearrangements in Oxytricha produce transcriptionally active extrachromosomal circular DNA
    Article Snippet: Paragraph title: 5′-Rapid amplification of cDNA ends (5′-RACE) ... Strand-specific, gene-specific primers were used to reverse transcribe 800 ng of DNase-treated total RNA using AMV reverse transcriptase according to manufacturer's instructions (NEB). cDNA was purified using MinElute (Qiagen) and 5 pmol of cDNA was terminal transferase-treated (NEB) to A-tail according to manufacturer's instructions.

    Article Title: DNA damage regulates direct association of TOR kinase with the RNA polymerase II–transcribed HMO1 gene
    Article Snippet: The cDNA was prepared from 500 ng total RNA using 1X AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 U of AMV reverse transcriptase (New England BioLabs) in a total reaction volume of 25 μl. .. The mixture was incubated at 42°C for 1 h. A ViiA 7 (Applied Biosystems) was used for quantitative PCR (qPCR) using Taq polymerase for amplification and SYBR Green I (Sigma) for detection.

    Article Title: Simian Rotaviruses Possess Divergent Gene Constellations That Originated from Interspecies Transmission and Reassortment ▿
    Article Snippet: .. Afterward, the dsRNA segments were resolved by electrophoresis on a 1% agarose gel, and each was recovered by using a QIAquick gel extraction kit (Qiagen). cDNAs were synthesized for each dsRNA segment using AMV reverse transcriptase (New England Biolabs) and then PCR amplified with ExTaq DNA polymerase (Takara). .. The cDNAs were cloned into the pGEM-T Easy vector (Promega).

    Article Title: Redox-Sensitive MarR Homologue BifR from Burkholderia thailandensis Regulates Biofilm Formation
    Article Snippet: .. The cDNA was prepared using 500 ng of total RNA. cDNA was made by mixing either EmrB, Bif R , Nudix , PhzF , or EcsC qPCR primers (EmrB_qpCR_Fw and EmrB_qpCR_Rev, BifR_qPCR_Fw, and BifR_qPCR_Rev, Nudix_qPCR_Fw and Nudix_qPCR_Rev, PhzF_qPCR_Fw and PhZF_qPCR_Rev, or EcsC_qPCR_Fw, and EcsC_qPCR_-Rev; ; gene-specific primers were used to increase sensitivity, since only specific transcripts will be reverse-transcribed) in 1× AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 units of AMV reverse transcriptase (New England Biolabs) in a total reaction volume of 25 μ L. The mixture was incubated at 42 °C for 1 h. A ViiA 7 (Applied Biosystems) was used for qPCR using Taq polymerase (New England Biolabs) for amplification and SYBR Green I (Sigma) for detection. .. For analysis of gene expression in WT, expression of emrB , bif R , nudix, ecsC , and phzF was normalized to the reference gene (glutamate synthase large subunit; BTH_I3014 , amplified using primers Glusynlg_qPCR_Fw and Glusynlg_qPCR_Rev) and reported as 2−ΔCT .

    Synthesized:

    Article Title: Simian Rotaviruses Possess Divergent Gene Constellations That Originated from Interspecies Transmission and Reassortment ▿
    Article Snippet: .. Afterward, the dsRNA segments were resolved by electrophoresis on a 1% agarose gel, and each was recovered by using a QIAquick gel extraction kit (Qiagen). cDNAs were synthesized for each dsRNA segment using AMV reverse transcriptase (New England Biolabs) and then PCR amplified with ExTaq DNA polymerase (Takara). .. The cDNAs were cloned into the pGEM-T Easy vector (Promega).

    Quantitative RT-PCR:

    Article Title: An in vivo selection method to optimize trans-splicing ribozymes
    Article Snippet: Paragraph title: Quantitative RT-PCR ... The transcriptions were incubated for 1 h at 42°C with AMV reverse transcriptase (NEB).

    Article Title: LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes
    Article Snippet: Paragraph title: qRT-PCR ... RNAs extracted from oocytes and RNAs coimmunoprecipitated with proteins were treated with Turbo DNase (Thermo Fisher Scientific), and then were reverse-transcribed into cDNA using a random hexamer primer and AMV Reverse Transcriptase (NEB). qPCR was performed using iTaq Universal SYBR Green Supermix or SsoAdvanced Universal SYBR Green Supermix on CFX96 (Biorad).

    Article Title: Gene Regulation by Redox-Sensitive Burkholderia thailandensis OhrR and Its Role in Bacterial Killing of Caenorhabditis elegans
    Article Snippet: Paragraph title: In vivo gene expression analysis using qRT-PCR. ... To this mixture, 8.5 μl of the master mix (containing 1× avian myeloblastosis virus [AMV] reverse transcriptase buffer, 1 mM MgCl2 , 1 mM deoxynucleoside triphosphate mix, 10 U AMV reverse transcriptase [New England BioLabs]) was added, making a total reaction volume of 25 μl, followed by incubation for 1 h at 42°C.

    SYBR Green Assay:

    Article Title: DNA damage regulates direct association of TOR kinase with the RNA polymerase II–transcribed HMO1 gene
    Article Snippet: The cDNA was prepared from 500 ng total RNA using 1X AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 U of AMV reverse transcriptase (New England BioLabs) in a total reaction volume of 25 μl. .. The mixture was incubated at 42°C for 1 h. A ViiA 7 (Applied Biosystems) was used for quantitative PCR (qPCR) using Taq polymerase for amplification and SYBR Green I (Sigma) for detection.

    Article Title: LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes
    Article Snippet: .. RNAs extracted from oocytes and RNAs coimmunoprecipitated with proteins were treated with Turbo DNase (Thermo Fisher Scientific), and then were reverse-transcribed into cDNA using a random hexamer primer and AMV Reverse Transcriptase (NEB). qPCR was performed using iTaq Universal SYBR Green Supermix or SsoAdvanced Universal SYBR Green Supermix on CFX96 (Biorad). .. In vitro Cyclin A protein degradation assay 6xHis-MBP-HRV3Csite-HA-Cyclin A protein was expressed using a modified pET plasmid vector in E. coli .

    Article Title: Redox-Sensitive MarR Homologue BifR from Burkholderia thailandensis Regulates Biofilm Formation
    Article Snippet: .. The cDNA was prepared using 500 ng of total RNA. cDNA was made by mixing either EmrB, Bif R , Nudix , PhzF , or EcsC qPCR primers (EmrB_qpCR_Fw and EmrB_qpCR_Rev, BifR_qPCR_Fw, and BifR_qPCR_Rev, Nudix_qPCR_Fw and Nudix_qPCR_Rev, PhzF_qPCR_Fw and PhZF_qPCR_Rev, or EcsC_qPCR_Fw, and EcsC_qPCR_-Rev; ; gene-specific primers were used to increase sensitivity, since only specific transcripts will be reverse-transcribed) in 1× AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 units of AMV reverse transcriptase (New England Biolabs) in a total reaction volume of 25 μ L. The mixture was incubated at 42 °C for 1 h. A ViiA 7 (Applied Biosystems) was used for qPCR using Taq polymerase (New England Biolabs) for amplification and SYBR Green I (Sigma) for detection. .. For analysis of gene expression in WT, expression of emrB , bif R , nudix, ecsC , and phzF was normalized to the reference gene (glutamate synthase large subunit; BTH_I3014 , amplified using primers Glusynlg_qPCR_Fw and Glusynlg_qPCR_Rev) and reported as 2−ΔCT .

    Incubation:

    Article Title: RsaC sRNA modulates the oxidative stress response of Staphylococcus aureus during manganese starvation
    Article Snippet: .. Primer extension assays Primer extension assays were performed using 30 μg of total RNA, incubated with 5′-radiolabeled oligonucleotide (PE-RsaC-Rev, ), 2.5 mM dNTPs and AMV reverse transcriptase (4 units, NEB). ..

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: Ten microliters of 2× reverse transcription mix (2× M-MuLV-Reverse Transcriptase reaction buffer, 0–200 units of M-MuLV-Reverse Transcriptase enzyme [New England Biolabs Inc.], or 10 units of AMV Reverse Transcriptase [New England Biolabs Inc.], 1 mM dNTPs [New England Biolabs Inc.], and 40 units of Murine RNase Inhibitor [New England Biolabs Inc.]) were added to the template mix. .. Reverse transcription comparing wild-type M-MuLV, AMV, SuperScript II, and SuperScript III used 200 U of M-MuLV or 10 U of AMV (New England Biolabs Inc.) as described above, or SuperScript II or III (Life Technologies) according to the manufacturer's suggestions with incubation temperatures of 42°C or 50°C, respectively.

    Article Title: RNA modification enzyme TruB is a tRNA chaperone
    Article Snippet: RNA from total extracts was reverse transcribed using AMV reverse transcriptase (New England Biolabs) according to the manufacturer’s protocol, using a TruB specific antisense primer that anneals to the 3′ end of the gene ( ). .. Briefly, total RNA (0.5 µg) was incubated with TruB antisense primer (∼50 µg) for 5 min at 65 °C, cooled, and added (5 µL) to the nuclease-free reaction mixture (20 µL) containing AMV reaction buffer (1×), AMV reverse transcriptase (4 U), and RNase inhibitor (8 U).

    Article Title: CsrA Represses Translation of sdiA, Which Encodes the N-Acylhomoserine-l-Lactone Receptor of Escherichia coli, by Binding Exclusively within the Coding Region of sdiA mRNA ▿ mRNA ▿ †
    Article Snippet: .. The reaction mixture was incubated for 15 min at 37°C following the addition of 1 U of AMV reverse transcriptase. .. Samples were heated for 2 min at 90°C prior to fractionation through 6% polyacrylamide sequencing gels.

    Article Title: An in vivo selection method to optimize trans-splicing ribozymes
    Article Snippet: .. The transcriptions were incubated for 1 h at 42°C with AMV reverse transcriptase (NEB). .. One-tenth of the reverse transcription reaction was used as template for quantitative PCR with the primers 35 and 36 for the substrate, and primers 37 and 38 for the product.

    Article Title: DNA damage regulates direct association of TOR kinase with the RNA polymerase II–transcribed HMO1 gene
    Article Snippet: The cDNA was prepared from 500 ng total RNA using 1X AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 U of AMV reverse transcriptase (New England BioLabs) in a total reaction volume of 25 μl. .. The mixture was incubated at 42°C for 1 h. A ViiA 7 (Applied Biosystems) was used for quantitative PCR (qPCR) using Taq polymerase for amplification and SYBR Green I (Sigma) for detection.

    Article Title: Gene Regulation by Redox-Sensitive Burkholderia thailandensis OhrR and Its Role in Bacterial Killing of Caenorhabditis elegans
    Article Snippet: .. To this mixture, 8.5 μl of the master mix (containing 1× avian myeloblastosis virus [AMV] reverse transcriptase buffer, 1 mM MgCl2 , 1 mM deoxynucleoside triphosphate mix, 10 U AMV reverse transcriptase [New England BioLabs]) was added, making a total reaction volume of 25 μl, followed by incubation for 1 h at 42°C. .. To compare differences in gene expression qualitatively, the cDNA was used as a template for PCR with gene-specific primers.

    Article Title: Redox-Sensitive MarR Homologue BifR from Burkholderia thailandensis Regulates Biofilm Formation
    Article Snippet: .. The cDNA was prepared using 500 ng of total RNA. cDNA was made by mixing either EmrB, Bif R , Nudix , PhzF , or EcsC qPCR primers (EmrB_qpCR_Fw and EmrB_qpCR_Rev, BifR_qPCR_Fw, and BifR_qPCR_Rev, Nudix_qPCR_Fw and Nudix_qPCR_Rev, PhzF_qPCR_Fw and PhZF_qPCR_Rev, or EcsC_qPCR_Fw, and EcsC_qPCR_-Rev; ; gene-specific primers were used to increase sensitivity, since only specific transcripts will be reverse-transcribed) in 1× AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 units of AMV reverse transcriptase (New England Biolabs) in a total reaction volume of 25 μ L. The mixture was incubated at 42 °C for 1 h. A ViiA 7 (Applied Biosystems) was used for qPCR using Taq polymerase (New England Biolabs) for amplification and SYBR Green I (Sigma) for detection. .. For analysis of gene expression in WT, expression of emrB , bif R , nudix, ecsC , and phzF was normalized to the reference gene (glutamate synthase large subunit; BTH_I3014 , amplified using primers Glusynlg_qPCR_Fw and Glusynlg_qPCR_Rev) and reported as 2−ΔCT .

    Expressing:

    Article Title: DNA damage regulates direct association of TOR kinase with the RNA polymerase II–transcribed HMO1 gene
    Article Snippet: Paragraph title: RNA isolation and in vivo gene expression ... The cDNA was prepared from 500 ng total RNA using 1X AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 U of AMV reverse transcriptase (New England BioLabs) in a total reaction volume of 25 μl.

    Article Title: Gene Regulation by Redox-Sensitive Burkholderia thailandensis OhrR and Its Role in Bacterial Killing of Caenorhabditis elegans
    Article Snippet: Paragraph title: In vivo gene expression analysis using qRT-PCR. ... To this mixture, 8.5 μl of the master mix (containing 1× avian myeloblastosis virus [AMV] reverse transcriptase buffer, 1 mM MgCl2 , 1 mM deoxynucleoside triphosphate mix, 10 U AMV reverse transcriptase [New England BioLabs]) was added, making a total reaction volume of 25 μl, followed by incubation for 1 h at 42°C.

    Article Title: Redox-Sensitive MarR Homologue BifR from Burkholderia thailandensis Regulates Biofilm Formation
    Article Snippet: Paragraph title: In Vivo Gene Expression and Operon Confirmation ... The cDNA was prepared using 500 ng of total RNA. cDNA was made by mixing either EmrB, Bif R , Nudix , PhzF , or EcsC qPCR primers (EmrB_qpCR_Fw and EmrB_qpCR_Rev, BifR_qPCR_Fw, and BifR_qPCR_Rev, Nudix_qPCR_Fw and Nudix_qPCR_Rev, PhzF_qPCR_Fw and PhZF_qPCR_Rev, or EcsC_qPCR_Fw, and EcsC_qPCR_-Rev; ; gene-specific primers were used to increase sensitivity, since only specific transcripts will be reverse-transcribed) in 1× AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 units of AMV reverse transcriptase (New England Biolabs) in a total reaction volume of 25 μ L. The mixture was incubated at 42 °C for 1 h. A ViiA 7 (Applied Biosystems) was used for qPCR using Taq polymerase (New England Biolabs) for amplification and SYBR Green I (Sigma) for detection.

    Transformation Assay:

    Article Title: Programmed genome rearrangements in Oxytricha produce transcriptionally active extrachromosomal circular DNA
    Article Snippet: Strand-specific, gene-specific primers were used to reverse transcribe 800 ng of DNase-treated total RNA using AMV reverse transcriptase according to manufacturer's instructions (NEB). cDNA was purified using MinElute (Qiagen) and 5 pmol of cDNA was terminal transferase-treated (NEB) to A-tail according to manufacturer's instructions. .. RACE products were gel extracted (Qiagen), transformed into One Shot TOP10 chemically competent cells (Invitrogen) and Sanger sequenced (Genewiz) to map the precise TSS in three validated eccDNAs.

    High Performance Liquid Chromatography:

    Article Title: Polymerase synthesis of four-base DNA from two stable dimeric nucleotides
    Article Snippet: Klenow fragment DNA Polymerase exo-, KlenTaq DNA Polymerase, Therminator™ DNA Polymerase, Vent® exo- DNA Polymerase, AMV Reverse Transcriptase, and dNTPs were purchased from NEB. .. High-performance liquid chromatography (HPLC) was performed using a system comprised of two Shimadzu LC-10AD pumps, SCL-10A controller, and SPD-M10A photodiode array detector.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: An in vivo selection method to optimize trans-splicing ribozymes
    Article Snippet: The transcriptions were incubated for 1 h at 42°C with AMV reverse transcriptase (NEB). .. The PCR was performed using the AB qPCR master mix on a Fast 7500 RT-PCR machine (Applied Biosystems).

    Sequencing:

    Article Title: RsaC sRNA modulates the oxidative stress response of Staphylococcus aureus during manganese starvation
    Article Snippet: Primer extension assays Primer extension assays were performed using 30 μg of total RNA, incubated with 5′-radiolabeled oligonucleotide (PE-RsaC-Rev, ), 2.5 mM dNTPs and AMV reverse transcriptase (4 units, NEB). .. Samples were finally precipitated and migrated on a denaturing 10% polyacrylamide gel, next to sequencing ladder.

    Article Title: Simian Rotaviruses Possess Divergent Gene Constellations That Originated from Interspecies Transmission and Reassortment ▿
    Article Snippet: Paragraph title: Sequencing of the complete genome of PTRV. ... Afterward, the dsRNA segments were resolved by electrophoresis on a 1% agarose gel, and each was recovered by using a QIAquick gel extraction kit (Qiagen). cDNAs were synthesized for each dsRNA segment using AMV reverse transcriptase (New England Biolabs) and then PCR amplified with ExTaq DNA polymerase (Takara).

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: .. The 3′ ligated product was then ligated to a mixed pool of equimolar amount of 5′ RNA adapters containing UMIs in 3 nt-blocks of random nucleotides and one of the two distinct consensus sequence sets (NNN-CGA-NNN-UAC-NNN and NNN-AUC-NNN-AGU-NNN) in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2 , 10 mM DTT, 1 mM ATP with T4 RNA ligase (Ambion, Foster City, CA, USA) at 25 °C for 2 h. The ligated product was precipitated with ethanol, and cDNA synthesis was performed using AMV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). cDNA was PCR-amplified with a common forward primer (5′–AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-3′) and a reverse primer containing 6 nt Illumina multiplexing barcode (5′–CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA–3′) using AccuPrime Pfx DNA polymerase (ThermoFisher, Waltham, MA, USA). .. Finally, the PCR product was purified from a 2% Certified Ultra Low Range agarose gel (Bio-Rad Laboratories, Hercules, CA, USA).

    Multiplexing:

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: .. The 3′ ligated product was then ligated to a mixed pool of equimolar amount of 5′ RNA adapters containing UMIs in 3 nt-blocks of random nucleotides and one of the two distinct consensus sequence sets (NNN-CGA-NNN-UAC-NNN and NNN-AUC-NNN-AGU-NNN) in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2 , 10 mM DTT, 1 mM ATP with T4 RNA ligase (Ambion, Foster City, CA, USA) at 25 °C for 2 h. The ligated product was precipitated with ethanol, and cDNA synthesis was performed using AMV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). cDNA was PCR-amplified with a common forward primer (5′–AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-3′) and a reverse primer containing 6 nt Illumina multiplexing barcode (5′–CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA–3′) using AccuPrime Pfx DNA polymerase (ThermoFisher, Waltham, MA, USA). .. Finally, the PCR product was purified from a 2% Certified Ultra Low Range agarose gel (Bio-Rad Laboratories, Hercules, CA, USA).

    In Vivo:

    Article Title: DNA damage regulates direct association of TOR kinase with the RNA polymerase II–transcribed HMO1 gene
    Article Snippet: Paragraph title: RNA isolation and in vivo gene expression ... The cDNA was prepared from 500 ng total RNA using 1X AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 U of AMV reverse transcriptase (New England BioLabs) in a total reaction volume of 25 μl.

    Article Title: Gene Regulation by Redox-Sensitive Burkholderia thailandensis OhrR and Its Role in Bacterial Killing of Caenorhabditis elegans
    Article Snippet: Paragraph title: In vivo gene expression analysis using qRT-PCR. ... To this mixture, 8.5 μl of the master mix (containing 1× avian myeloblastosis virus [AMV] reverse transcriptase buffer, 1 mM MgCl2 , 1 mM deoxynucleoside triphosphate mix, 10 U AMV reverse transcriptase [New England BioLabs]) was added, making a total reaction volume of 25 μl, followed by incubation for 1 h at 42°C.

    Article Title: Redox-Sensitive MarR Homologue BifR from Burkholderia thailandensis Regulates Biofilm Formation
    Article Snippet: Paragraph title: In Vivo Gene Expression and Operon Confirmation ... The cDNA was prepared using 500 ng of total RNA. cDNA was made by mixing either EmrB, Bif R , Nudix , PhzF , or EcsC qPCR primers (EmrB_qpCR_Fw and EmrB_qpCR_Rev, BifR_qPCR_Fw, and BifR_qPCR_Rev, Nudix_qPCR_Fw and Nudix_qPCR_Rev, PhzF_qPCR_Fw and PhZF_qPCR_Rev, or EcsC_qPCR_Fw, and EcsC_qPCR_-Rev; ; gene-specific primers were used to increase sensitivity, since only specific transcripts will be reverse-transcribed) in 1× AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 units of AMV reverse transcriptase (New England Biolabs) in a total reaction volume of 25 μ L. The mixture was incubated at 42 °C for 1 h. A ViiA 7 (Applied Biosystems) was used for qPCR using Taq polymerase (New England Biolabs) for amplification and SYBR Green I (Sigma) for detection.

    RNA Sequencing Assay:

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: Paragraph title: Small RNA-seq library construction ... The 3′ ligated product was then ligated to a mixed pool of equimolar amount of 5′ RNA adapters containing UMIs in 3 nt-blocks of random nucleotides and one of the two distinct consensus sequence sets (NNN-CGA-NNN-UAC-NNN and NNN-AUC-NNN-AGU-NNN) in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2 , 10 mM DTT, 1 mM ATP with T4 RNA ligase (Ambion, Foster City, CA, USA) at 25 °C for 2 h. The ligated product was precipitated with ethanol, and cDNA synthesis was performed using AMV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). cDNA was PCR-amplified with a common forward primer (5′–AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-3′) and a reverse primer containing 6 nt Illumina multiplexing barcode (5′–CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA–3′) using AccuPrime Pfx DNA polymerase (ThermoFisher, Waltham, MA, USA).

    Isolation:

    Article Title: An in vivo selection method to optimize trans-splicing ribozymes
    Article Snippet: Total RNA was isolated from logarithmically growing E. coli cells after induction with 1 mM IPTG, using the RNeasy kit (Qiagen). .. The transcriptions were incubated for 1 h at 42°C with AMV reverse transcriptase (NEB).

    Article Title: DNA damage regulates direct association of TOR kinase with the RNA polymerase II–transcribed HMO1 gene
    Article Snippet: Paragraph title: RNA isolation and in vivo gene expression ... The cDNA was prepared from 500 ng total RNA using 1X AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 U of AMV reverse transcriptase (New England BioLabs) in a total reaction volume of 25 μl.

    Article Title: Redox-Sensitive MarR Homologue BifR from Burkholderia thailandensis Regulates Biofilm Formation
    Article Snippet: Total RNA was isolated using the illustra RNAspin Mini Isolation kit (GE Healthcare). .. The cDNA was prepared using 500 ng of total RNA. cDNA was made by mixing either EmrB, Bif R , Nudix , PhzF , or EcsC qPCR primers (EmrB_qpCR_Fw and EmrB_qpCR_Rev, BifR_qPCR_Fw, and BifR_qPCR_Rev, Nudix_qPCR_Fw and Nudix_qPCR_Rev, PhzF_qPCR_Fw and PhZF_qPCR_Rev, or EcsC_qPCR_Fw, and EcsC_qPCR_-Rev; ; gene-specific primers were used to increase sensitivity, since only specific transcripts will be reverse-transcribed) in 1× AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 units of AMV reverse transcriptase (New England Biolabs) in a total reaction volume of 25 μ L. The mixture was incubated at 42 °C for 1 h. A ViiA 7 (Applied Biosystems) was used for qPCR using Taq polymerase (New England Biolabs) for amplification and SYBR Green I (Sigma) for detection.

    Size-exclusion Chromatography:

    Article Title: An in vivo selection method to optimize trans-splicing ribozymes
    Article Snippet: The transcriptions were incubated for 1 h at 42°C with AMV reverse transcriptase (NEB). .. Each PCR cycle included incubations of 30 sec at 95°C, 30 sec at 57°C, and 30 sec at 72°C.

    Labeling:

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: Reverse transcription of a synthetic RNA-DNA (5′-P-rArGrCrArGrUrGrGrCrUrGrGrUrUrGrArGrArUrUrUCTGTAGGCACCATCAAT-3′) was performed as follows: 10 pmol of template and 20 pmol of 5′-end IRDye 700 labeled reverse transcription primers 5′-IRDye 700-ATTGATGGTGCCTACAG-3′, or 5′-IRDye 700-ATTGATGGTGCCTA-3′ in a final volume of 10 μL were denatured for 5 min at 90°C and placed on ice. .. Ten microliters of 2× reverse transcription mix (2× M-MuLV-Reverse Transcriptase reaction buffer, 0–200 units of M-MuLV-Reverse Transcriptase enzyme [New England Biolabs Inc.], or 10 units of AMV Reverse Transcriptase [New England Biolabs Inc.], 1 mM dNTPs [New England Biolabs Inc.], and 40 units of Murine RNase Inhibitor [New England Biolabs Inc.]) were added to the template mix.

    Titration:

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: Ten microliters of 2× reverse transcription mix (2× M-MuLV-Reverse Transcriptase reaction buffer, 0–200 units of M-MuLV-Reverse Transcriptase enzyme [New England Biolabs Inc.], or 10 units of AMV Reverse Transcriptase [New England Biolabs Inc.], 1 mM dNTPs [New England Biolabs Inc.], and 40 units of Murine RNase Inhibitor [New England Biolabs Inc.]) were added to the template mix. .. For dNTP titration reactions, 1 mM free Mg2+ was maintained by increasing the concentration of MgCl2 proportionally with the increased dNTPs.

    Polymerase Chain Reaction:

    Article Title: RsaC sRNA modulates the oxidative stress response of Staphylococcus aureus during manganese starvation
    Article Snippet: Primer extension assays Primer extension assays were performed using 30 μg of total RNA, incubated with 5′-radiolabeled oligonucleotide (PE-RsaC-Rev, ), 2.5 mM dNTPs and AMV reverse transcriptase (4 units, NEB). .. The sequencing ladder was obtained with a DNA template (PCR with oligonucleotides PE-RsaC-For and PE-RsaC-Rev, from genomic DNA).

    Article Title: RNA modification enzyme TruB is a tRNA chaperone
    Article Snippet: Paragraph title: Reverse Transcription Followed by PCR. ... RNA from total extracts was reverse transcribed using AMV reverse transcriptase (New England Biolabs) according to the manufacturer’s protocol, using a TruB specific antisense primer that anneals to the 3′ end of the gene ( ).

    Article Title: An in vivo selection method to optimize trans-splicing ribozymes
    Article Snippet: The transcriptions were incubated for 1 h at 42°C with AMV reverse transcriptase (NEB). .. The PCR was performed using the AB qPCR master mix on a Fast 7500 RT-PCR machine (Applied Biosystems).

    Article Title: DNA damage regulates direct association of TOR kinase with the RNA polymerase II–transcribed HMO1 gene
    Article Snippet: Contaminating DNA was removed using Turbo DNase (Ambion), and absence of DNA was verified by PCR. .. The cDNA was prepared from 500 ng total RNA using 1X AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 U of AMV reverse transcriptase (New England BioLabs) in a total reaction volume of 25 μl.

    Article Title: Simian Rotaviruses Possess Divergent Gene Constellations That Originated from Interspecies Transmission and Reassortment ▿
    Article Snippet: .. Afterward, the dsRNA segments were resolved by electrophoresis on a 1% agarose gel, and each was recovered by using a QIAquick gel extraction kit (Qiagen). cDNAs were synthesized for each dsRNA segment using AMV reverse transcriptase (New England Biolabs) and then PCR amplified with ExTaq DNA polymerase (Takara). .. The cDNAs were cloned into the pGEM-T Easy vector (Promega).

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: .. The 3′ ligated product was then ligated to a mixed pool of equimolar amount of 5′ RNA adapters containing UMIs in 3 nt-blocks of random nucleotides and one of the two distinct consensus sequence sets (NNN-CGA-NNN-UAC-NNN and NNN-AUC-NNN-AGU-NNN) in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2 , 10 mM DTT, 1 mM ATP with T4 RNA ligase (Ambion, Foster City, CA, USA) at 25 °C for 2 h. The ligated product was precipitated with ethanol, and cDNA synthesis was performed using AMV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). cDNA was PCR-amplified with a common forward primer (5′–AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-3′) and a reverse primer containing 6 nt Illumina multiplexing barcode (5′–CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA–3′) using AccuPrime Pfx DNA polymerase (ThermoFisher, Waltham, MA, USA). .. Finally, the PCR product was purified from a 2% Certified Ultra Low Range agarose gel (Bio-Rad Laboratories, Hercules, CA, USA).

    Article Title: Gene Regulation by Redox-Sensitive Burkholderia thailandensis OhrR and Its Role in Bacterial Killing of Caenorhabditis elegans
    Article Snippet: To this mixture, 8.5 μl of the master mix (containing 1× avian myeloblastosis virus [AMV] reverse transcriptase buffer, 1 mM MgCl2 , 1 mM deoxynucleoside triphosphate mix, 10 U AMV reverse transcriptase [New England BioLabs]) was added, making a total reaction volume of 25 μl, followed by incubation for 1 h at 42°C. .. To compare differences in gene expression qualitatively, the cDNA was used as a template for PCR with gene-specific primers.

    Article Title: Redox-Sensitive MarR Homologue BifR from Burkholderia thailandensis Regulates Biofilm Formation
    Article Snippet: DNA contamination was removed using Turbo DNase (Ambion), and the absence of DNA was verified by PCR. .. The cDNA was prepared using 500 ng of total RNA. cDNA was made by mixing either EmrB, Bif R , Nudix , PhzF , or EcsC qPCR primers (EmrB_qpCR_Fw and EmrB_qpCR_Rev, BifR_qPCR_Fw, and BifR_qPCR_Rev, Nudix_qPCR_Fw and Nudix_qPCR_Rev, PhzF_qPCR_Fw and PhZF_qPCR_Rev, or EcsC_qPCR_Fw, and EcsC_qPCR_-Rev; ; gene-specific primers were used to increase sensitivity, since only specific transcripts will be reverse-transcribed) in 1× AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 units of AMV reverse transcriptase (New England Biolabs) in a total reaction volume of 25 μ L. The mixture was incubated at 42 °C for 1 h. A ViiA 7 (Applied Biosystems) was used for qPCR using Taq polymerase (New England Biolabs) for amplification and SYBR Green I (Sigma) for detection.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: Ten microliters of 2× reverse transcription mix (2× M-MuLV-Reverse Transcriptase reaction buffer, 0–200 units of M-MuLV-Reverse Transcriptase enzyme [New England Biolabs Inc.], or 10 units of AMV Reverse Transcriptase [New England Biolabs Inc.], 1 mM dNTPs [New England Biolabs Inc.], and 40 units of Murine RNase Inhibitor [New England Biolabs Inc.]) were added to the template mix. .. The reactions were quenched with 2× RNA denaturing loading buffer and separated by denaturing PAGE.

    Gel Extraction:

    Article Title: Simian Rotaviruses Possess Divergent Gene Constellations That Originated from Interspecies Transmission and Reassortment ▿
    Article Snippet: .. Afterward, the dsRNA segments were resolved by electrophoresis on a 1% agarose gel, and each was recovered by using a QIAquick gel extraction kit (Qiagen). cDNAs were synthesized for each dsRNA segment using AMV reverse transcriptase (New England Biolabs) and then PCR amplified with ExTaq DNA polymerase (Takara). .. The cDNAs were cloned into the pGEM-T Easy vector (Promega).

    Nested PCR:

    Article Title: Programmed genome rearrangements in Oxytricha produce transcriptionally active extrachromosomal circular DNA
    Article Snippet: Strand-specific, gene-specific primers were used to reverse transcribe 800 ng of DNase-treated total RNA using AMV reverse transcriptase according to manufacturer's instructions (NEB). cDNA was purified using MinElute (Qiagen) and 5 pmol of cDNA was terminal transferase-treated (NEB) to A-tail according to manufacturer's instructions. .. The A-tailed cDNA was amplified using 40 net cycles of nested PCR before resolving the products on an agarose gel.

    Activated Clotting Time Assay:

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: .. The 3′ ligated product was then ligated to a mixed pool of equimolar amount of 5′ RNA adapters containing UMIs in 3 nt-blocks of random nucleotides and one of the two distinct consensus sequence sets (NNN-CGA-NNN-UAC-NNN and NNN-AUC-NNN-AGU-NNN) in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2 , 10 mM DTT, 1 mM ATP with T4 RNA ligase (Ambion, Foster City, CA, USA) at 25 °C for 2 h. The ligated product was precipitated with ethanol, and cDNA synthesis was performed using AMV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). cDNA was PCR-amplified with a common forward primer (5′–AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-3′) and a reverse primer containing 6 nt Illumina multiplexing barcode (5′–CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA–3′) using AccuPrime Pfx DNA polymerase (ThermoFisher, Waltham, MA, USA). .. Finally, the PCR product was purified from a 2% Certified Ultra Low Range agarose gel (Bio-Rad Laboratories, Hercules, CA, USA).

    Purification:

    Article Title: Programmed genome rearrangements in Oxytricha produce transcriptionally active extrachromosomal circular DNA
    Article Snippet: .. Strand-specific, gene-specific primers were used to reverse transcribe 800 ng of DNase-treated total RNA using AMV reverse transcriptase according to manufacturer's instructions (NEB). cDNA was purified using MinElute (Qiagen) and 5 pmol of cDNA was terminal transferase-treated (NEB) to A-tail according to manufacturer's instructions. .. The A-tailed cDNA was amplified using 40 net cycles of nested PCR before resolving the products on an agarose gel.

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: The 54–71 nt (18–35 nt small RNA + 36 nt 3′ UMI adapter) 3′ ligated product was purified from a 10% denaturing urea-polyacrylamide gel. .. The 3′ ligated product was then ligated to a mixed pool of equimolar amount of 5′ RNA adapters containing UMIs in 3 nt-blocks of random nucleotides and one of the two distinct consensus sequence sets (NNN-CGA-NNN-UAC-NNN and NNN-AUC-NNN-AGU-NNN) in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2 , 10 mM DTT, 1 mM ATP with T4 RNA ligase (Ambion, Foster City, CA, USA) at 25 °C for 2 h. The ligated product was precipitated with ethanol, and cDNA synthesis was performed using AMV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). cDNA was PCR-amplified with a common forward primer (5′–AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-3′) and a reverse primer containing 6 nt Illumina multiplexing barcode (5′–CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA–3′) using AccuPrime Pfx DNA polymerase (ThermoFisher, Waltham, MA, USA).

    Plasmid Preparation:

    Article Title: Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes
    Article Snippet: Klenow large fragment, Therminator™ II, M-MuLV (RNase H− ) reverse transcriptase, AMV reverse transcriptase, RNase inhibitor and λ-exonuclease were purchased from New England Biolabs. .. Plasmid HydGdCTD5 (5.176 kb,PCR template) was provided by Professor Peter Roach at Southampton University School of Chemistry.

    Article Title: Simian Rotaviruses Possess Divergent Gene Constellations That Originated from Interspecies Transmission and Reassortment ▿
    Article Snippet: Afterward, the dsRNA segments were resolved by electrophoresis on a 1% agarose gel, and each was recovered by using a QIAquick gel extraction kit (Qiagen). cDNAs were synthesized for each dsRNA segment using AMV reverse transcriptase (New England Biolabs) and then PCR amplified with ExTaq DNA polymerase (Takara). .. The cDNAs were cloned into the pGEM-T Easy vector (Promega).

    Real-time Polymerase Chain Reaction:

    Article Title: An in vivo selection method to optimize trans-splicing ribozymes
    Article Snippet: The transcriptions were incubated for 1 h at 42°C with AMV reverse transcriptase (NEB). .. One-tenth of the reverse transcription reaction was used as template for quantitative PCR with the primers 35 and 36 for the substrate, and primers 37 and 38 for the product.

    Article Title: DNA damage regulates direct association of TOR kinase with the RNA polymerase II–transcribed HMO1 gene
    Article Snippet: The cDNA was prepared from 500 ng total RNA using 1X AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 U of AMV reverse transcriptase (New England BioLabs) in a total reaction volume of 25 μl. .. The mixture was incubated at 42°C for 1 h. A ViiA 7 (Applied Biosystems) was used for quantitative PCR (qPCR) using Taq polymerase for amplification and SYBR Green I (Sigma) for detection.

    Article Title: LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes
    Article Snippet: .. RNAs extracted from oocytes and RNAs coimmunoprecipitated with proteins were treated with Turbo DNase (Thermo Fisher Scientific), and then were reverse-transcribed into cDNA using a random hexamer primer and AMV Reverse Transcriptase (NEB). qPCR was performed using iTaq Universal SYBR Green Supermix or SsoAdvanced Universal SYBR Green Supermix on CFX96 (Biorad). .. In vitro Cyclin A protein degradation assay 6xHis-MBP-HRV3Csite-HA-Cyclin A protein was expressed using a modified pET plasmid vector in E. coli .

    Article Title: Redox-Sensitive MarR Homologue BifR from Burkholderia thailandensis Regulates Biofilm Formation
    Article Snippet: .. The cDNA was prepared using 500 ng of total RNA. cDNA was made by mixing either EmrB, Bif R , Nudix , PhzF , or EcsC qPCR primers (EmrB_qpCR_Fw and EmrB_qpCR_Rev, BifR_qPCR_Fw, and BifR_qPCR_Rev, Nudix_qPCR_Fw and Nudix_qPCR_Rev, PhzF_qPCR_Fw and PhZF_qPCR_Rev, or EcsC_qPCR_Fw, and EcsC_qPCR_-Rev; ; gene-specific primers were used to increase sensitivity, since only specific transcripts will be reverse-transcribed) in 1× AMV reverse transcriptase buffer with 1 mM MgCl2 , 1 mM dNTP, and 10 units of AMV reverse transcriptase (New England Biolabs) in a total reaction volume of 25 μ L. The mixture was incubated at 42 °C for 1 h. A ViiA 7 (Applied Biosystems) was used for qPCR using Taq polymerase (New England Biolabs) for amplification and SYBR Green I (Sigma) for detection. .. For analysis of gene expression in WT, expression of emrB , bif R , nudix, ecsC , and phzF was normalized to the reference gene (glutamate synthase large subunit; BTH_I3014 , amplified using primers Glusynlg_qPCR_Fw and Glusynlg_qPCR_Rev) and reported as 2−ΔCT .

    Agarose Gel Electrophoresis:

    Article Title: Programmed genome rearrangements in Oxytricha produce transcriptionally active extrachromosomal circular DNA
    Article Snippet: Strand-specific, gene-specific primers were used to reverse transcribe 800 ng of DNase-treated total RNA using AMV reverse transcriptase according to manufacturer's instructions (NEB). cDNA was purified using MinElute (Qiagen) and 5 pmol of cDNA was terminal transferase-treated (NEB) to A-tail according to manufacturer's instructions. .. The A-tailed cDNA was amplified using 40 net cycles of nested PCR before resolving the products on an agarose gel.

    Article Title: Simian Rotaviruses Possess Divergent Gene Constellations That Originated from Interspecies Transmission and Reassortment ▿
    Article Snippet: .. Afterward, the dsRNA segments were resolved by electrophoresis on a 1% agarose gel, and each was recovered by using a QIAquick gel extraction kit (Qiagen). cDNAs were synthesized for each dsRNA segment using AMV reverse transcriptase (New England Biolabs) and then PCR amplified with ExTaq DNA polymerase (Takara). .. The cDNAs were cloned into the pGEM-T Easy vector (Promega).

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: The 3′ ligated product was then ligated to a mixed pool of equimolar amount of 5′ RNA adapters containing UMIs in 3 nt-blocks of random nucleotides and one of the two distinct consensus sequence sets (NNN-CGA-NNN-UAC-NNN and NNN-AUC-NNN-AGU-NNN) in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2 , 10 mM DTT, 1 mM ATP with T4 RNA ligase (Ambion, Foster City, CA, USA) at 25 °C for 2 h. The ligated product was precipitated with ethanol, and cDNA synthesis was performed using AMV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). cDNA was PCR-amplified with a common forward primer (5′–AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-3′) and a reverse primer containing 6 nt Illumina multiplexing barcode (5′–CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA–3′) using AccuPrime Pfx DNA polymerase (ThermoFisher, Waltham, MA, USA). .. Finally, the PCR product was purified from a 2% Certified Ultra Low Range agarose gel (Bio-Rad Laboratories, Hercules, CA, USA).

    Article Title: Gene Regulation by Redox-Sensitive Burkholderia thailandensis OhrR and Its Role in Bacterial Killing of Caenorhabditis elegans
    Article Snippet: To this mixture, 8.5 μl of the master mix (containing 1× avian myeloblastosis virus [AMV] reverse transcriptase buffer, 1 mM MgCl2 , 1 mM deoxynucleoside triphosphate mix, 10 U AMV reverse transcriptase [New England BioLabs]) was added, making a total reaction volume of 25 μl, followed by incubation for 1 h at 42°C. .. PCR products were electrophoresed on a 1.2% agarose gel and observed after staining with ethidium bromide.

    Electrophoresis:

    Article Title: Simian Rotaviruses Possess Divergent Gene Constellations That Originated from Interspecies Transmission and Reassortment ▿
    Article Snippet: .. Afterward, the dsRNA segments were resolved by electrophoresis on a 1% agarose gel, and each was recovered by using a QIAquick gel extraction kit (Qiagen). cDNAs were synthesized for each dsRNA segment using AMV reverse transcriptase (New England Biolabs) and then PCR amplified with ExTaq DNA polymerase (Takara). .. The cDNAs were cloned into the pGEM-T Easy vector (Promega).

    Random Hexamer Labeling:

    Article Title: LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes
    Article Snippet: .. RNAs extracted from oocytes and RNAs coimmunoprecipitated with proteins were treated with Turbo DNase (Thermo Fisher Scientific), and then were reverse-transcribed into cDNA using a random hexamer primer and AMV Reverse Transcriptase (NEB). qPCR was performed using iTaq Universal SYBR Green Supermix or SsoAdvanced Universal SYBR Green Supermix on CFX96 (Biorad). .. In vitro Cyclin A protein degradation assay 6xHis-MBP-HRV3Csite-HA-Cyclin A protein was expressed using a modified pET plasmid vector in E. coli .

    Concentration Assay:

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA
    Article Snippet: Ten microliters of 2× reverse transcription mix (2× M-MuLV-Reverse Transcriptase reaction buffer, 0–200 units of M-MuLV-Reverse Transcriptase enzyme [New England Biolabs Inc.], or 10 units of AMV Reverse Transcriptase [New England Biolabs Inc.], 1 mM dNTPs [New England Biolabs Inc.], and 40 units of Murine RNase Inhibitor [New England Biolabs Inc.]) were added to the template mix. .. For dNTP titration reactions, 1 mM free Mg2+ was maintained by increasing the concentration of MgCl2 proportionally with the increased dNTPs.

    Staining:

    Article Title: Gene Regulation by Redox-Sensitive Burkholderia thailandensis OhrR and Its Role in Bacterial Killing of Caenorhabditis elegans
    Article Snippet: To this mixture, 8.5 μl of the master mix (containing 1× avian myeloblastosis virus [AMV] reverse transcriptase buffer, 1 mM MgCl2 , 1 mM deoxynucleoside triphosphate mix, 10 U AMV reverse transcriptase [New England BioLabs]) was added, making a total reaction volume of 25 μl, followed by incubation for 1 h at 42°C. .. PCR products were electrophoresed on a 1.2% agarose gel and observed after staining with ethidium bromide.

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    New England Biolabs amv reverse transcriptase
    Reverse transcription using AHP dUTP. ( A ) RNA template T5 with primer P3. ( B and C ) Twenty percent denaturing PAGE analysis of reactions using <t>AMV</t> and <t>M-MuLV</t> (RNase H-) reverse transcriptases at 42°C for 15 h.
    Amv Reverse Transcriptase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Reverse transcription using AHP dUTP. ( A ) RNA template T5 with primer P3. ( B and C ) Twenty percent denaturing PAGE analysis of reactions using AMV and M-MuLV (RNase H-) reverse transcriptases at 42°C for 15 h.

    Journal: Nucleic Acids Research

    Article Title: Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

    doi: 10.1093/nar/gkw028

    Figure Lengend Snippet: Reverse transcription using AHP dUTP. ( A ) RNA template T5 with primer P3. ( B and C ) Twenty percent denaturing PAGE analysis of reactions using AMV and M-MuLV (RNase H-) reverse transcriptases at 42°C for 15 h.

    Article Snippet: Klenow large fragment, Therminator™ II, M-MuLV (RNase H− ) reverse transcriptase, AMV reverse transcriptase, RNase inhibitor and λ-exonuclease were purchased from New England Biolabs.

    Techniques: Polyacrylamide Gel Electrophoresis

    Identification of RNase III cleavage sites in betT mRNA in vitro and in vivo . (A) In vitro cleavage of the full-length synthetic betT mRNA. The 5′-end-labeled betT transcript (4 pmol) was incubated with purified RNase III (1 pmol) in cleavage buffer with (+) or without (-) MgCl 2 at 37°C. The size of the cleavage products was estimated using size markers generated by internally labeled transcripts. The major cleavage products are indicated with arrows. Other minor cleavage products are indicated with asterisks. (B) Primer extension analysis of betT mRNA. Total RNA was prepared from MG1655 rnc harboring pBetRS1 and either pKAN6B or pRNC1, which exogenously overexpressed betT mRNA, hybridized with a 5′- 32 P-end-labeled primer (betT+120R), and extended using AMV reverse transcriptase. Sequencing ladders were produced using the same primer used in cDNA synthesis and PCR DNA, encompassing the betT gene as a template. (C) The predicted secondary structure of betT mRNA region encompassing RNase III cleavage sites. The secondary structure was determined using the M-fold program [33] . (D) In vitro cleavage of the model hairpin RNA of betT mRNA. 3′-end-labeled betT model hairpin (25 pmol) was incubated with purified RNase III (1 pmol) in a cleavage buffer with (+) or without (−) MgCl 2 , respectively. Cleavage products (I, II, III, and IV) were identified using size markers generated by alkaline hydrolysis and RNase T1 digestion. Relative abundance of each cleavage product was assessed by measuring the radioactivity of each band and plotted.

    Journal: PLoS ONE

    Article Title: Two Tandem RNase III Cleavage Sites Determine betT mRNA Stability in Response to Osmotic Stress in Escherichia coli

    doi: 10.1371/journal.pone.0100520

    Figure Lengend Snippet: Identification of RNase III cleavage sites in betT mRNA in vitro and in vivo . (A) In vitro cleavage of the full-length synthetic betT mRNA. The 5′-end-labeled betT transcript (4 pmol) was incubated with purified RNase III (1 pmol) in cleavage buffer with (+) or without (-) MgCl 2 at 37°C. The size of the cleavage products was estimated using size markers generated by internally labeled transcripts. The major cleavage products are indicated with arrows. Other minor cleavage products are indicated with asterisks. (B) Primer extension analysis of betT mRNA. Total RNA was prepared from MG1655 rnc harboring pBetRS1 and either pKAN6B or pRNC1, which exogenously overexpressed betT mRNA, hybridized with a 5′- 32 P-end-labeled primer (betT+120R), and extended using AMV reverse transcriptase. Sequencing ladders were produced using the same primer used in cDNA synthesis and PCR DNA, encompassing the betT gene as a template. (C) The predicted secondary structure of betT mRNA region encompassing RNase III cleavage sites. The secondary structure was determined using the M-fold program [33] . (D) In vitro cleavage of the model hairpin RNA of betT mRNA. 3′-end-labeled betT model hairpin (25 pmol) was incubated with purified RNase III (1 pmol) in a cleavage buffer with (+) or without (−) MgCl 2 , respectively. Cleavage products (I, II, III, and IV) were identified using size markers generated by alkaline hydrolysis and RNase T1 digestion. Relative abundance of each cleavage product was assessed by measuring the radioactivity of each band and plotted.

    Article Snippet: Total RNA was hybridized with a 5′-32 P-end-labeled primer (betT+273R) and extended using AMV reverse transcriptase.

    Techniques: In Vitro, In Vivo, Labeling, Incubation, Purification, Generated, Sequencing, Produced, Polymerase Chain Reaction, Radioactivity