标签归档：hiscribe

HiScribe® SP6 RNA Synthesis Kit |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

The HiScribe SP6 RNA Synthesis Kit is designed for the in vitro transcription of RNA using SP6 RNA Polymerase. This kit is suitable for synthesis of high yield RNA transcripts and for incorporation of cap analogs (not included) or modified nucleotides (not included) to obtain capped, biotin-labeled or dye-labeled RNA. The kit is also capable of synthesizing high specific activity radiolabeled RNA for use as probes or targets.

RNA synthesized from this kit is suitable for many applications including RNA structure and function studies, ribozyme biochemistry, probes for RNase protection or gel shift assays, hybridization-based blots, anti-sense RNA or RNAi experiments, microarray analysis, microinjection, sgRNA synthesis and in vitro translation studies.

This kit contains sufficient reagents for 50 reactions of 25 µl each. Each standard reaction yields ≥ 80 µg of RNA from 1 µg SP6 Control Template DNA. Each kit can yield ≥ 4 mg of RNA.

Figure 1: Transcription by SP6 RNA Polymerase.
HiScribe® SP6 RNA Synthesis Kit |
Figure 2: Comparison of RNA yields from commercially-available SP6 in vitro transcription kits demonstrates the robustness of the NEB HiScribe SP6 RNA Synthesis Kit
HiScribe® SP6 RNA Synthesis Kit |
Reactions with 1 μg linearized plasmid DNA templates were incubated at 37°C in a PCR machine according to the manufacturer’s recommendations. Transcripts were treated with DNase I, purified by LiCl precipitation and quantified using a NanoDrop™ Spectrophotometer. The HiScribe SP6 RNA Synthesis Kit from NEB is faster and achieves higher yields for RNAs of various lengths.
产品类别:
RNA Capping,
RNA Synthesis In vitro Transcription (IVT)

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • E2070S     -20    
        SP6 Reaction Buffer B2070AVIAL -20 1 x 0.125 ml Not Applicable
        SP6 RNA Polymerase Mix M2070AVIAL -20 1 x 0.125 ml Not Applicable
        SP6 Control Template N2070AVIAL -20 1 x 0.02 ml 0.25 mg/ml
        ATP (Tris) N2071AVIAL -20 1 x 0.125 ml 50 mM
        GTP (Tris) N2072AVIAL -20 1 x 0.125 ml 50 mM
        UTP (Tris) N2073AVIAL -20 1 x 0.125 ml 50 mM
        CTP (Tris) N2074AVIAL -20 1 x 0.125 ml 50 mM
        DNase I (RNase-free) M0303AVIAL -20 1 x 0.1 ml 2,000 units/ml
        LiCl Solution B2051AVIAL -20 1 x 1.4 ml Not Applicable
        Dithiothreitol (DTT) B1222AVIAL -20 1 x 0.5 ml 100 mM

  • 特性和用法

    需要但不提供的材料

    DNA Template: The DNA template must be linear and contain the SP6 RNA Polymerase promoter with the correct orientation in relation to the target sequence to be transcribed.
    General: Thermocycler or 37°C dry air incubator, microcentrifuge, nuclease-free water, nuclease-free tubes and tips
    Purification: Phenol, chloroform, ethanol, 3 M Sodium Acetate, pH 5.2 or 5 M Ammonium Acetate, spin columns, equipment for RNA quantitation
    Gel Analysis: Gels, running buffers, loading dye, nucleic acid ladders, gel apparatus, power supply

    Optional Materials
    Cap Analogs: NEB #S1411, #S1404, #S1405, #S1406, #S1407
    Modified NTPs: Biotin-, Fluorescein-, Digoxigenin-, Aminoallyl-
    Labeling: [α-32P] labeled ribonucleotide (800-6,000 Ci/mmol), storage phosphor screen

  • 相关产品

    相关产品

    • e2065-hiscribe-t7-arca-mrna-kit
    • e2060-hiscribe-t7-arca-mrna-kit-with-tailing
    • e2040-hiscribe-t7-high-yield-rna-synthesis-kit
    • e2050-hiscribe-t7-quick-high-yield-rna-synthesis-kit
    • 2X RNA 上样染料
    • m0307-rnase-inhibitor-human-placenta
    • 小鼠 RNase 抑制剂
    • m0303-dnase-i-rnase-free
    • Q5® 热启动超保真 DNA 聚合酶
    • Monarch® PCR & DNA 纯化试剂盒（5 μg）
    • ssRNA Ladder
    • 低分子量 ssRNA Ladder
    • s1411-3-o-me-m7g5ppp5g-rna-cap-structure-analog
    • s1405-m7g5ppp5a-rna-cap-structure-analog
    • s1406-g5ppp5a-rna-cap-structure-analog
    • s1407-g5ppp5g-rna-cap-structure-analog
    • s1404-m7g5ppp5g-rna-cap-structure-analog
    • m2080-vaccinia-capping-system
    • mRNA 帽结构 2-O-甲基转移酶
    • E. coli Poly(A) 聚合酶
    • rNTP 套装
    • T3 RNA 聚合酶
    • m0251-t7-rna-polymerase
    • SP6 RNA 聚合酶

操作说明、说明书 & 用法

  • 操作说明

    1. RNA Synthesis Protocols (E2070)
    2. Purification of Synthesized RNA (E2070)

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE2070

  • 应用实例

    • Scaling of High-Yield In vitro Transcription Reactions for Linear Increase of RNA Production

FAQs & 问题解决指南

  • FAQs

    1. What is the promoter sequence for SP6 RNA Polymerase?
    2. Does transcription with SP6 RNA Polymerase require a primer?
    3. Does SP6 RNA Polymerase leave an extra base at the end of the transcript?
    4. Will SP6 RNA Polymerase initiate transcription from a single-stranded DNA template?
    5. Will SP6 RNA Polymerase work on an uncut plasmid?
    6. Can aberrant RNA be produced using SP6 RNA polymerase?
    7. Can kit components from the HiScribe SP6 RNA Synthesis Kit be substituted with SP6 RNA Polymerase and Reaction Buffer (NEB #M0207)?
    8. What is the sequence of the SP6 Control Template?
    9. Can I use the Monarch RNA Cleanup Kits to cleanup my in vitro transcription (IVT) reaction?
    10. Are modified nucleotides included in the kit?
    11. Do I need to add DTT to the reaction?

  • 问题解决指南

    Control Reaction
    The SP6 control template is a linearized plasmid containing the Cypridina luciferase (CLuc) gene under the transcriptional control of the SP6 promoter. The size of the run-off transcript is 1.76 kb. The control reaction should yield ≥ 80 µg of RNA in 2 hours.

    If the control reaction is not working, there may be technical problems during the reaction set up. Repeat the reaction by following the protocol carefully; take every precaution to avoid RNase contamination. Contact NEB for technical assistance.

    The control plasmid pCLuc-SP6 sequence can be found within the DNA Sequences and Maps Tool. The pCLuc-SP6 control template is generated by linearizing the plasmid with XbaI.

    Low Yield of Full-length RNA
    If the transcription reaction with your template generates full-length RNA, but the yield is significantly lower than expected, it is possible that contaminants in the DNA template are inhibiting the RNA polymerase, or the DNA concentration may be incorrect. Alternatively, additional purification of DNA template may be required. Phenol:chloroform extraction is recommended (see template DNA preparation section).

    Low Yield of Short Transcript
    High yields of short transcripts (< 0.3 kb) are achieved by extending incubation time and increasing the amount of template. Incubation of reactions up to 16 hours (overnight) or using up to 2 μg of template will help to achieve maximum yield.

    RNA Transcript Smearing on Denaturing Gel
    If the RNA appears degraded (e.g. smeared) on a denaturing agarose or polyacrylamide gel, the DNA template is most likely contaminated with RNase.  DNA templates contaminated with RNAse can affect the length and yield of RNA synthesized (a smear below the expected transcript length). If the plasmid DNA template is contaminated with RNase, perform phenol:chloroform extraction followed by ethanol precipitation. Dissolve the DNA in nuclease-free water (see template DNA preparation section).
     
    RNA Transcript of Larger Size than Expected
    If the RNA transcript appears larger than expected on a denaturing gel, template plasmid DNA may be incompletely digested. Even small amounts of circular DNA can produce large amounts of long transcripts. Check the template for complete digestion on an agarose gel compared to an uncut plasmid sample. If undigested plasmid is confirmed, repeat the restriction enzyme digestion.
    Larger size bands may also be observed when the RNA transcript is not completely denatured due to the presence of strong secondary structure.

    RNA Transcript of Smaller Size than Expected
    If denaturing gel analysis shows the presence of smaller bands than expected, it is most likely due to premature termination by the polymerase. Some sequences which resemble the SP6 RNA Polymerase termination signal may cause premature termination. For GC-rich templates, or templates with secondary structure, incubation at 42°C may improve the yield of full-length transcript.
    If premature termination of transcription is found in high specific activity radiolabeled RNA probe synthesis, increase the concentration of the “limiting nucleotide”. Additional unlabeled limiting NTP can be added to the reaction to increase the proportion of full-length transcript, however the improvement in yield of full-length product will compromise the specific activity of the probe.

HiScribe® T7 ARCA mRNA Kit |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

Most eukaryotic mRNAs require a 7-methyl guanosine (m7G) cap structure at the 5´ end and a Poly(A) tail at the 3´ end to be efficiently translated. By using a DNA template encoding a poly(A) tail, the HiScribe T7 ARCA mRNA Kit can be used to synthesize capped and tailed mRNAs. The cap structure is added to the mRNA by co-transcriptional incorporation of Anti-Reverse Cap Analog (ARCA) (NEB #S1411) using T7 RNA Polymerase. Poly(A) tail is incorporated during the transcription reaction. The kit also includes DNase I and LiCl for DNA template removal and quick mRNA purification.

Additionally, the kit is capable of partial incorporation of modified UTP and CTP (up to 50% each) without affecting the mRNA yield significantly. By using a DNA template encoding a poly(A) tail, capped and tailed modified mRNA can be synthesized in a single reaction in 30 minutes. mRNAs synthesized with the kit can be used for cell transfection, microinjection, in vitro translation and RNA vaccines.

ARCA is incorporated into mRNA exclusively in the correct orientation, generating capped mRNA that is more efficiently translated. Standard cap analogs can be incorporated in either direction resulting in only 50% of capped mRNA that is functional in protein translation.

Figure 1. Structure of Anti-Reverse Cap Analog (ARCA, NEB #S1411)HiScribe® T7 ARCA mRNA Kit |
Methylation at the 3´ position of 7mG forces the cap structure to be attached to mRNA in the correct orientation.
Figure 2. Overview of mRNA synthesis work flow with the HiScribe T7 ARCA mRNA KitHiScribe® T7 ARCA mRNA Kit |
产品类别:
RNA Capping,
RNA Synthesis In vitro Transcription (IVT)

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • E2065S     -20    
        T7 RNA Polymerase Mix M0255AAVIAL -20 1 x 0.04 ml Not Applicable
        ARCA/NTP Mix N2053AVIAL -20 1 x 0.2 ml Not Applicable
        DNase I (RNase-free) M0303AAVIAL -20 1 x 0.04 ml 2,000 units/ml
        CLuc Control Template N0247AVIAL -20 1 x 20 µl 0.25 mg/ml
        LiCl Solution B2051AVIAL -20 1 x 1.4 ml Not Applicable
        Dithiothreitol (DTT) B1222AVIAL -20 1 x 0.5 ml 100 mM

  • 特性和用法

    需要但不提供的材料

    • DNA template
    • Thermocycler or 37°C incubator.
    • Nuclease-free water
    • Buffer- or water-saturated phenol:chloroform
    • Ethanol
    • 3 M Sodium acetate, pH 5.2
    • 5 M Ammonium acetate
    • Spin columns (see Monarch® RNA Cleanup Kits, NEB #T2040 or #T2050)
    • Gels, running buffers and gel box
    • Equipment for RNA analysis

  • 相关产品

    相关产品

    • e2040-hiscribe-t7-high-yield-rna-synthesis-kit
    • e2050-hiscribe-t7-quick-high-yield-rna-synthesis-kit
    • e2060-hiscribe-t7-arca-mrna-kit-with-tailing
    • 2X RNA 上样染料
    • m0307-rnase-inhibitor-human-placenta
    • 小鼠 RNase 抑制剂
    • m0303-dnase-i-rnase-free
    • Q5® 热启动超保真 DNA 聚合酶
    • ssRNA Ladder
    • 低分子量 ssRNA Ladder
    • s1411-3-o-me-m7g5ppp5g-rna-cap-structure-analog
    • s1405-m7g5ppp5a-rna-cap-structure-analog
    • s1406-g5ppp5a-rna-cap-structure-analog
    • s1407-g5ppp5g-rna-cap-structure-analog
    • s1404-m7g5ppp5g-rna-cap-structure-analog
    • m2080-vaccinia-capping-system
    • mRNA 帽结构 2-O-甲基转移酶
    • E. coli Poly(A) 聚合酶
    • rNTP 混合液
    • rNTP 套装
    • T2040 Monarch RNA Cleanup Kit 50 ug
    • Monarch® PCR & DNA 纯化试剂盒（5 μg）

操作说明、说明书 & 用法

  • 操作说明

    1. Standard mRNA Synthesis (E2065)
    2. mRNA Synthesis with Modified Nucleotides (E2065)
    3. mRNA Purification (E2065)
    4. Evaluation of Reaction Products (E2065)

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE2065

  • 应用实例

    • Scaling of High-Yield In vitro Transcription Reactions for Linear Increase of RNA Production

FAQs & 问题解决指南

  • FAQs

    1. HiScribe&reg; T7 ARCA mRNA Kit (with tailing) What is the difference between the HiScribe T7 ARCA mRNA Kit (NEB #E2065) and the HiScribe T7 ARCA mRNA Kit (with Tailing)(NEB #E2060)?
    2. I currently use MessageMAX™ T7 ARCA-capped Message Transcription Kit, which mRNA synthesis kit from NEB should I use?
    3. I currently use mMessage mMachine® T7 Ultra Transcription Kit, which mRNA synthesis kit from NEB should I use?
    4. Can modified nucleotides be used with the HiScribe T7 ARCA mRNA kits?
    5. What is the difference between the HiScribe T7 ARCA mRNA kits and the HiScribe T7 High Yield RNA Synthesis Kit (E2040) and HiScribe T7 Quick RNA Synthesis Kit (E2050)?
    6. Can I use the Monarch RNA Cleanup Kits to cleanup my in vitro transcription (IVT) reaction?
    7. How can I improve on a low yield of RNA from the transcription reaction?
    8. Are modified nucleotides included in the kit?
    9. Do I need to add DTT to the reaction?

  • 问题解决指南

    Control Reaction
    The CLuc control template DNA is a linearized plasmid containing the Cypridina luciferase gene under the transcriptional control of the T7 promoter. The size of the runoff transcript is 1.6 kb. The control reaction should yield ≥ 15 μg RNA transcript in 30 minutes.

    If the control reaction is not working, there may be technical problems during reaction set up. Repeat the reaction by following the protocol carefully; take all precautions to avoid RNase contamination. Contact NEB for technical assistance. The control plasmid sequence can be found at www.neb.com.

    The CLuc control template is generated by linearizing the plasmid with restriction enzyme XbaI.

    Low Yield of Full-length RNA
    If the transcription reaction with your template generates full-length RNA, but the yield is significantly lower than expected, it is possible that contaminants in the DNA template are inhibiting the RNA polymerase, or the DNA concentration may be incorrect. Alternatively, additional purification of DNA template may be required. Phenol:chloroform extraction is recommended (see template DNA preparation section).

    Low Yield of Short Transcript
    High yields of short transcripts (< 0.3 kb) are achieved by extending incubation time and increasing the amount of template. Incubation of reactions up to 16 hours (overnight) or using up to 2 μg of template will help to achieve maximum yield. Alternatively, clean up the DNA template using a spin column based method, Monarch PCR & DNA Cleanup Kit (5 μg), NEB #T1030.

    RNA Transcript Smearing on Denaturing
    Gel If the RNA appears degraded (e.g., smeared) on denaturing agarose or polyacrylamide gel, the DNA template is likely contaminated with RNase. DNA templates contaminated with RNase can affect the length and yield of RNA synthesized (a smear below the expected transcript length). If the plasmid DNA template is contaminated with RNase, perform phenol:chloroform extraction, then ethanol precipitate and dissolve the DNA in nuclease-free water (see template DNA preparation section).

    RNA Transcript of Larger Size than Expected
    If the RNA transcript appears larger than expected on a denaturing gel, plasmid DNA may be incompletely digested. Even small amounts of undigested circular plasmid DNA can produce large amounts of long transcripts. Check template for complete digestion. If undigested plasmid is confirmed, repeat restriction enzyme digestion.

    Larger size bands may also be observed when the RNA transcript is not completely denatured due to the presence of strong secondary structure.

    RNA Transcript of Smaller Size than Expected
    If denaturing gel analysis shows the presence of smaller bands than the expected size, it is most likely due to premature termination by the polymerase. Sequences with resemblance to T7 RNA Polymerase termination signals will cause premature termination. Incubating the transcription reaction at lower temperatures, for example at 30°C, may increase the proportion of full-length transcript, however the yield will be decreased. For GC rich templates, or templates with secondary structures, incubation at 42°C may improve yield of full-length transcript.

    Tailing Length Control Tail length is defined by the poly(A) coding length on the DNA template. Poly(A) tails longer than 125 nt have minimal effect on enhancing mRNA function.

    mRNA not Functional

    • Verify the mRNA is intact, capped and tailed.
    • Be sure the mRNA is clean, free from any inhibitors of downstream experiments.
    • Follow instructions carefully with appropriate controls.
    • Verify the DNA template has the correct sequence.

HiScribe® T7 ARCA mRNA Kit (with tailing) |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

Most eukaryotic mRNAs require a 7-methyl guanosine (m7G) cap structure at the 5´ end and a poly(A) tail at the 3´ end to be efficiently translated. The HiScribe T7 ARCA mRNA Kit (with tailing) is designed for quick production of ARCA capped and poly(A) tailed mRNA in vitro. Capped mRNAs are synthesized by co-transcriptional incorporation of Anti-Reverse Cap Analog (ARCA, NEB #S1411) using T7 RNA Polymerase. The transcription reaction can be set up easily by combining the ARCA/NTP mix, T7 RNA Polymerase Mix and a suitable DNA template. The kit also allows for partial incorporation of 5mCTP, Pseudo-UTP and other modified nucleotides into mRNA. After a brief DNase I treatment to remove the template DNA, capped mRNA is poly(A) tailed with Poly(A) Polymerase. mRNAs synthesized with the kit can be used for cell transfection, microinjection, in vitro translation and RNA vaccines.

ARCA is incorporated into mRNA exclusively in the correct orientation, generating capped mRNA that is more efficiently translated. Standard cap analogs can be incorporated in either direction resulting in only 50% of capped mRNA that is functional in protein translation.

Figure 1. Structure of Anti-Reverse Cap Analog (ARCA, NEB #S1411)HiScribe® T7 ARCA mRNA Kit (with tailing) |
Methylation at the 3´ position of 7mG forces the cap structure to be attached to mRNA in the correct orientation.
Figure 2. Overview of mRNA synthesis work flow with the HiScribe T7 ARCA mRNA Kit (with tailing)HiScribe® T7 ARCA mRNA Kit (with tailing) |
产品类别:
RNA Capping,
RNA Synthesis In vitro Transcription (IVT)

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • E2060S     -20    
        Poly(A) Polymerase Reaction Buffer B0276SVIAL -20 1 x 1.5 ml 10 X
        T7 RNA Polymerase Mix M0255AAVIAL -20 1 x 0.04 ml Not Applicable
        ARCA/NTP Mix N2053AVIAL -20 1 x 0.2 ml Not Applicable
        DNase I (RNase-free) M0303AAVIAL -20 1 x 0.04 ml 2,000 units/ml
        CLuc Control Template N0247AVIAL -20 1 x 20 µl 0.25 mg/ml
        LiCl Solution B2051AVIAL -20 1 x 1.4 ml Not Applicable
        E. coli Poly(A) Polymerase M0444AVIAL -20 1 x 0.1 ml Not Applicable
        Dithiothreitol (DTT) B1222AVIAL -20 1 x 0.5 ml 100 mM

  • 特性和用法

    需要但不提供的材料

    • DNA template
    • Thermocycler or 37°C incubator.
    • Nuclease-free water
    • Buffer- or water-saturated phenol:chloroform
    • Ethanol
    • 3 M Sodium acetate, pH 5.2
    • 5 M Ammonium acetate
    • Spin columns (see Monarch® RNA Cleanup Kits, NEB #T2040 or #T2050)
    • Gels, running buffers and gel box
    • Equipment for RNA analysis

  • 相关产品

    相关产品

    • e2040-hiscribe-t7-high-yield-rna-synthesis-kit
    • e2050-hiscribe-t7-quick-high-yield-rna-synthesis-kit
    • e2065-hiscribe-t7-arca-mrna-kit
    • 2X RNA 上样染料
    • m0307-rnase-inhibitor-human-placenta
    • 小鼠 RNase 抑制剂
    • m0303-dnase-i-rnase-free
    • Q5® 热启动超保真 DNA 聚合酶
    • ssRNA Ladder
    • 低分子量 ssRNA Ladder
    • s1411-3-o-me-m7g5ppp5g-rna-cap-structure-analog
    • s1405-m7g5ppp5a-rna-cap-structure-analog
    • s1406-g5ppp5a-rna-cap-structure-analog
    • s1407-g5ppp5g-rna-cap-structure-analog
    • s1404-m7g5ppp5g-rna-cap-structure-analog
    • m2080-vaccinia-capping-system
    • mRNA 帽结构 2-O-甲基转移酶
    • E. coli Poly(A) 聚合酶
    • rNTP 混合液
    • rNTP 套装
    • Monarch® PCR & DNA 纯化试剂盒（5 μg）
    • T2040 Monarch RNA Cleanup Kit 50 ug

  • 注意事项

    1. 试剂盒所有组分都应贮存于 –20℃。试剂盒提供的试剂足够进行每次 20 μl 的 20 次反应。每次标准反应 1 μg 对照模板可合成高达 20 μg 的加帽 mRNA。Poly(A) 加尾和 LiCl 沉淀纯化后，可获得高达 25 μg 的加帽和加尾 mRNA。

操作说明、说明书 & 用法

  • 操作说明

    1. Standard mRNA Synthesis (E2060)
    2. mRNA Synthesis with Modified Nucleotides (E2060)
    3. mRNA Purification (E2060)
    4. Evaluation of Reaction Products (E2060)

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE2060

  • 应用实例

    • Scaling of High-Yield In vitro Transcription Reactions for Linear Increase of RNA Production

FAQs & 问题解决指南

  • FAQs

    1. HiScribe&reg; T7 ARCA mRNA Kit (with tailing) What is the difference between the HiScribe T7 ARCA mRNA Kit (NEB #E2065) and the HiScribe T7 ARCA mRNA Kit (with Tailing)(NEB #E2060)?
    2. I currently use mMessage mMachine® T7 Ultra Transcription Kit, which mRNA synthesis kit from NEB should I use?
    3. I currently use MessageMAX™ T7 ARCA-capped Message Transcription Kit, which mRNA synthesis kit from NEB should I use?
    4. Can modified nucleotides be used with the HiScribe T7 ARCA mRNA kits?
    5. What is the difference between the HiScribe T7 ARCA mRNA kits and the HiScribe T7 High Yield RNA Synthesis Kit (E2040) and HiScribe T7 Quick RNA Synthesis Kit (E2050)?
    6. Can I use the Monarch RNA Cleanup Kits to cleanup my in vitro transcription (IVT) reaction?
    7. How can I improve on a low yield of RNA from the transcription reaction?
    8. Are modified nucleotides included in the kit?
    9. Do I need to add DTT to the reaction?

  • 问题解决指南

    • Control Reaction
      The CLuc control template DNA is a linearized plasmid containing the Cypridina luciferase gene under the transcriptional control of the T7 promoter. The size of the runoff transcript is 1.6 kb. The control reaction should yield ≥ 15 μg RNA transcript in 30 minutes.

      If the control reaction is not working, there may be technical problems during reaction set up. Repeat the reaction by following the protocol carefully; take all precautions to avoid RNase contamination. Contact NEB for technical assistance.

      The control plasmid sequence can be found here. The CLuc control template is generated by linearizing the plasmid with restriction enzyme Xba I.

    • Low Yield of Full-length RNA
      If the transcription reaction with your template generates full-length RNA, but the yield is significantly lower than expected, it is possible that contaminants in the DNA template are inhibiting the RNA polymerase, or the DNA concentration may be incorrect. Alternatively, additional purification of DNA template may be required. Phenol:chloroform extraction is recommended (see template DNA preparation section). 
    • Low Yield of Short Transcript
      High yields of short transcripts (< 0.3 kb) are achieved by extending incubation time and increasing the amount of template. Incubation of reactions up to 16 hours (overnight) or using up to 2 μg of template will help to achieve maximum yield. Alternatively, clean up the DNA template using a spin column
      based method, Monarch PCR & DNA Cleanup Kit (5 μg), NEB #T1030.
    • RNA Transcript Smearing on Denaturing Gel
      If the RNA appears degraded (e.g., smeared) on denaturing agarose or polyacrylamide gel, the DNA template is likely contaminated with RNase. DNA templates contaminated with RNase can affect the length and yield of RNA synthesized (a smear below the expected transcript length). We recommend evaluating the plasmid DNA template with the RNase Contamination assay Kit (NEB #E3320). If the plasmid DNA template is contaminated with RNase, perform phenol:chloroform extraction, then ethanol precipitate and dissolve the DNA in nuclease-free water. If the plasmid DNA template is contaminated with RNase, perform phenol:chloroform extraction, then ethanol precipitate and dissolve the DNA in nuclease-free water (see template DNA preparation section). 
    • RNA Transcript of Larger Size than Expected
      If the RNA transcript appears larger than expected on a denaturing gel, plasmid DNA may be incompletely digested. Even small amounts of undigested circular plasmid DNA can produce large amounts of long transcripts. Check template for complete digestion. If undigested plasmid is confirmed, repeat restriction enzyme digestion.

      Larger size bands may also be observed when the RNA transcript is not completely denatured due to the presence of strong secondary structures.

    • RNA Transcript of Smaller Size than Expected
      If denaturing gel analysis shows the presence of smaller bands than the expected size, it is most likely due to premature termination by the polymerase. Sequences with resemblance to T7 RNA Polymerase termination signals will cause premature termination. Incubating the transcription reaction at lower temperatures, for example at 30°C, may increase the proportion of full-length transcript, however the yield will be decreased. For GC rich templates, or templates with secondary structures, incubation at 42°C may improve yield of full-length transcript.
    • Tailing Length Control
      A standard 30 min tailing reaction can add a poly(A) tail at least 150 nt in length to an average size mRNA generated from the IVT reaction. Short RNA may require longer incubation time for sufficient tailing.
    • No Tailing or Partial Tailing
      3′ end of the mRNA must be exposed for efficient tailing. Because T7 RNA
      Polymerase tends to generate 3′ end heterogeneity by adding extra bases, a
      small percentage of the mRNA may adopt alternate structures which may not
      be suitable for tailing. The following tips may help with successful tailing.
      • Run the whole mRNA synthesis work flow without freezing the RNA
        between steps.
      • To avoid preferential tailing, pre-incubate tailing mix at 37°C for 3 minutes
        before adding Poly(A) Polymerase. Mix well immediately.
      • Tailing reaction should be at 37–40°C. Lower temperatures are not
        recommended.
      • If still no tailing, redesign the DNA template with different sequences at
        the 3′ end.
    • mRNA not functional
      • Verify the mRNA is intact, capped and tailed.
      • Be sure the mRNA is clean, free from any inhibitors of downstream experiments.
      • Follow instructions carefully with appropriate controls.
      • Verify the DNA template has the correct sequence.

HiScribe® T7 High Yield RNA Synthesis Kit |NEB酶试剂 New England Biolabs

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产品信息

The HiScribe T7 High Yield RNA Synthesis Kit is an extremely flexible system for in vitro transcription of RNA using T7 RNA Polymerase. The kit allows for synthesis many kinds of RNA including internally labeled and co-transcriptionally capped transcripts.

RNA synthesized from the kit is suitable for many applications including RNA structure and function studies, ribozyme biochemistry, probes for RNase protection assays and hybridization based blots, anti-sense RNA and RNAi experiments, microarray analysis, microinjection, and in vitro translation and RNA vaccines.

The kit contains sufficient reagents for 50 reactions of 20 μl each. Each standard reaction yields up to 180 μg of RNA from 1 μg control template. Each kit can yield up to 9 mg RNA. For 32P labeling, the kit contains enough reagents for 100 reactions of 20 μl each. 

Materials Not Included:

  • DNA Template: The DNA template must be linear and contain the T7 RNA Polymerase promoter with correct orientation in relation to target sequence to be transcribed. 
  • 3′-O-Me-m7G(5′)ppp(5′)G RNA Cap Structure Analog (NEB #S1411)
  • m7G(5′)ppp(5′)G RNA Cap Structure Analog (NEB #S1404)
  • m7G(5′)ppp(5′)A RNA Cap Structure Analog (NEB #S1405)
  • G(5′)ppp(5′)A RNA Cap Structure Analog (NEB #S1406)
  • G(5′)ppp(5′)G RNA Cap Structure Analog (NEB #S1407)
  • Modified-NTP: Biotin-, Fluorescein-, Digoxigenin-, or Aminoallyl-NTP 
  • Labeling: [α-32P] labeled ribonucleotide (800-6,000 Ci/mmol) 
  • General: 37°C incubator or PCR machine, nuclease-free water 
  • DNase I: DNase I (RNase-free) (NEB #M0303) 
  • Purification: Buffer- or water-saturated phenol/chloroform, ethanol and 3 M sodium acetate, pH 5.2, spin columns 
  • Gel Analysis: Gels and running buffers, gel apparatus, power supply

Figure 1. Transcription by T7 RNA Polymerase HiScribe® T7 High Yield RNA Synthesis Kit |
Figure 2. Time course of standard RNA synthesis from three DNA templates HiScribe® T7 High Yield RNA Synthesis Kit |
Reactions were incubated at 37°C in a PCR machine. Transcripts were purified by spin columns and quantified on NanoDrop™ Spectrophotometer.
Figure 3. Effect of template amount on RNA yield HiScribe® T7 High Yield RNA Synthesis Kit |
Standard reactions were incubated at 37°Cin a PCR machine for 2 hours. Transcripts were purified by spin columns and quantified on NanoDrop™ Spectrophotometer.
Figure 4: Improved RNA yield and integrity from extended duration transcription reactions HiScribe® T7 High Yield RNA Synthesis Kit |
reactions were assembled, in duplicate, according to the manufacturers’ suggested protocols using 3 ng of dsDNA template encoding a 1.8 kb RNA, and incubated at 37°C for 16, 24 and 40 hours. At each time point, the corresponding tubes were transferred to -20°C to stop the reaction. Transcription reactions were column purified after the last time point.

(A) Transcript yield – After column purification, RNA concentration was measured using a NanoDrop spectrophotometer and total RNA yield was calculated. These data demonstrate that a substantially higher yield of RNA was synthesized using the HiScribe T7 High Yield RNA Synthesis Kit as compared to the competitor’s kit.

(B) Transcript integrity – 150 ng of column purified RNA was run a 1.2% denaturing agarose gel, stained with ethidium bromide and visualized by UV fluorescence. The data demonstrate greatly improved transcript integrity after extended duration RNA synthesis reactions using the HiScribe T7 High Yield RNA Synthesis Kit as compared to the competitor’s kit.

产品类别:
RNA Capping,
RNA Synthesis In vitro Transcription (IVT)

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • E2040S     -20    
        CTP  N0454AVIAL -20 1 x 0.1 ml 100 mM
        FLuc Control Template N0426AVIAL -20 1 x 0.01 ml 0.5 µg/µl
        UTP  N0453AVIAL -20 1 x 0.1 ml 100 mM
        ATP N0451AVIAL -20 1 x 0.1 ml 100 mM
        T7 RNA Polymerase Mix M0255AVIAL -20 1 x 0.1 ml Not Applicable
        GTP  N0452AVIAL -20 1 x 0.1 ml 100 mM
        10X T7 Reaction Buffer B2041AVIAL -20 1 x 0.1 ml Not Applicable
        Dithiothreitol (DTT) B1222AVIAL -20 1 x 0.5 ml 100 mM

  • 相关产品

    相关产品

    • 2X RNA 上样染料
    • m0307-rnase-inhibitor-human-placenta
    • 小鼠 RNase 抑制剂
    • m0303-dnase-i-rnase-free
    • Q5® 热启动超保真 DNA 聚合酶
    • ssRNA Ladder
    • 低分子量 ssRNA Ladder
    • s1411-3-o-me-m7g5ppp5g-rna-cap-structure-analog
    • s1405-m7g5ppp5a-rna-cap-structure-analog
    • s1406-g5ppp5a-rna-cap-structure-analog
    • s1407-g5ppp5g-rna-cap-structure-analog
    • s1404-m7g5ppp5g-rna-cap-structure-analog
    • m2080-vaccinia-capping-system
    • mRNA 帽结构 2-O-甲基转移酶
    • E. coli Poly(A) 聚合酶
    • rNTP 混合液
    • rNTP 套装

操作说明、说明书 & 用法

  • 操作说明

    1. DNA Template Preparation (E2040)
    2. RNA Synthesis with Modified Nucleotides (E2040)
    3. Purification of Synthesized RNA (E2040)
    4. Standard RNA Synthesis (E2040)
    5. Capped RNA Synthesis (E2040)
    6. High Specific Activity Radiolabeled RNA Probe Synthesis (E2040)
    7. Evaluation of Reaction Products (E2040)
    8. Poly(A) Tailing of RNA using E. coli Poly(A) Polymerase (NEB# M0276)
    9. Protocol for Co-transcriptional capping using CleanCap® Reagent AG from TriLink and HiScribe™ T7 High Yield RNA Synthesis Kit from New England Biolabs®

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE2040

  • 应用实例

    • Scaling of High-Yield In vitro Transcription Reactions for Linear Increase of RNA Production

FAQs & 问题解决指南

  • FAQs

    1. Can I use the Monarch RNA Cleanup Kits to cleanup my in vitro transcription (IVT) reaction?
    2. How can I improve on a low yield of RNA from the transcription reaction?
    3. Are modified nucleotides included in the kit?
    4. Do I need to add DTT to the reaction?

  • 问题解决指南

    Control Reaction

    The FLuc control template DNA is a linearized plasmid containing the firefly luciferase gene under the transcriptional control of T7 promoter. The size of the runoff transcript is 1.8 kb. The control reaction should yield ≥ 150 μg RNA transcript in 2 hours.

    If the control reaction is not working, there may be technical problems during reaction set up. Repeat the reaction by following the protocol carefully; take any precaution to avoid RNase contamination. Contact NEB for technical assistance.

    The control plasmid sequence can be found within the DNA Sequences and Maps Tool under the name “FLuc Control Plasmid”. The FLuc control template is generated by linearizing the plasmid with StuI.

    Low Yield of Full-length RNA

    If the transcription reaction with your template generates full-length RNA, but the yield is significantly lower than expected, it is possible that contaminants in the DNA template are inhibiting the RNA polymerase, or the DNA concentration may be incorrect. Alternatively, additional purification of DNA template may be required. Phenol-chloroform extraction is recommended (see template DNA preparation section).

    Low Yield of Short Transcript

    High yields of short transcripts (< 0.3 kb) are achieved by extending incubation time and increasing the amount of template. Incubation of reactions up to 16 hours (overnight) or using up to 2 μg of template will help to achieve maximum yield.

    RNA Transcript Smearing on Denaturing Gel

    If the RNA appears degraded (e.g. smeared) on denaturing agarose or polyacrylamide gel, DNA template is contaminated with RNase. DNA templates contaminated with RNase can affect the length and yield of RNA synthesized (a smear below the expected transcript length). If the plasmid DNA template is contaminated with RNase, perform phenol/chloroform extraction, then ethanol precipitate and dissolve the DNA in nuclease-free water (see template DNA preparation section).

    RNA Transcript of Larger Size than Expected

    If the RNA transcript appears larger than expected on a denaturing gel, template plasmid DNA may be incompletely digested. Even small amounts of undigested circular DNA can produce large amounts of long transcripts. Check template for complete digestion, if undigested plasmid is confirmed, repeat restriction enzyme digestion.

    Larger size bands may also be observed when the RNA transcript is not completely denatured due to the presence of strong secondary structures.

    RNA Transcript of Smaller Size than Expected

    If denaturing gel analysis shows the presence of smaller bands than the expected size, it is most likely due to premature termination by the polymerase. Some sequences which resemble T7 RNA Polymerase termination signals will cause premature termination. Incubating the transcription reaction at lower temperatures, for example at 30°C, may increase the proportion of full-length transcript, however the yield will be decreased. For GC rich templates, or templates with secondary structures, incubation at 42°C may improve yield of full-length transcript.

    If premature termination of transcription is found in high specific activity radiolabeled RNA probe synthesis, increase the concentration of “limiting NTP”. Additional “cold” NTP can be added to the reaction to increase the proportion of full-length transcript, however the improvement in yield of full-length product will compromise the specific activity of the probe.

     

  • 实验技巧

    It is important to mix each component well before setting up reactions. 

    Make sure reactions are thoroughly mixed.

    We recommend incubating the reactions in a dry air incubator or in a PCR machine.