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View additional product information for SuperScript™ II Reverse Transcriptase - FAQs (18064071, 18064022, 18064014)
22 product FAQs found
如果在不含反转录酶的对照管/孔中生成扩增产物，必须从RNA样品中去除残留的基因组DNA。您可通过下述实验方案从总RNA中去除基因组DNA。
在冰上，将以下成分添加到无菌的 0.5 mL微量离心管中：
1.总RNA，最好小于或等于1 μg。（见下文注1。）
2.1.0 μL的10 X DNase缓冲液 (200 mM Tris, pH 8.3, 500 mM KCl, 20 mM MgCl2)。
3.0.1 U–3.0 U的DNase I（无RNase，货号18047019）或1.0 U的扩增级Dnase I,（货号18068015。参见下文注2。）
4.加入DEPC处理的水，将总体积加至10 μL。
5.在室温下孵育15分钟。（参见下文注3。）
6.加入1 μL 25 mM EDTA终止反应，并在65°C下加热10分钟。（参见下文注4。）
7.置于冰上1分钟。
8.通过短暂的离心处理进行回收。这样产生的混合物可直接用于反转录反应。
请留意以下注意事项：
1.如需处理更多的RNA，可线性放大整个反应体系。在10 μL的反应体系中，RNA不要超过2 μg。加入的RNA过多会导致溶液粘度增加，从而抑制DNase I扩散并找到DNA。
2.扩增级DNase I已经过充分纯化，去除了其他所谓的“RNase-free”酶制备过程后仍然残留的痕量核糖核酸酶的活性，不需要添加RNase抑制剂。
3.务必保证孵育时间不超过15分钟、温度不高于室温。温度过高或者孵育时间过长会导致RNA出现Mg2+依赖性水解。
4.这一步骤需要小心地吸取所有溶液，以确保二价的金属阳离子(Mg2+)的浓度能够得到有效控制。
5.由于DNase I必须加热到65°C来灭活酶，在加入EDAT之后，必须保证游离的二价金属离子浓度足够低（少于1 mM），以免RNA出现化学水解。另请参见下文的参考文献。
在加入EDTA后， Mg2+:EDTA的摩尔比例约为1:1。EDTA会与Mg2+分子按照1:1的摩尔比例进行螯合。因此，该RNA可直接被用于下游的反转录反应。加入第一链反转录酶缓冲液会使镁离子的终浓度为2.5mM。如果反转录反应缓冲液中不含MgCl2，则可向反应系统中添加MgCl2直至其终浓度达到2.5 mM。这样MgCl2的净终浓度约为2.25-2.5 mM。
RNA水解参考文献：
Molekulyarnaya Biologiya (1987) 21:1235-1241.
References on the mechanism of hydrolysis by other cations:
Eichorn GL and Butzov JY (1965) Biopolymers 3:79.
Butzov JY and Eichorn GL (1965) Biopolymers 3:95.
Farkas WR (1968) Biochim Biophys Acta 155:401.
第一篇论文的作者表达了这样一种观点，即由于阳离子引起的非特异性水解（通过2'，3' 磷酸环化进行）类似于RNA剪接时发生的特异性水解。
cDNA合成所需的RNA模板量具有高度的灵活性，取决于可用的样品量和个人需求。通常情况下，1 μg总RNA可进行一次常规20-μL反转录反应。
有些人觉得，反转录之后形成的RNA:DNA双链之中的RNA会抑制PCR引物退火和扩增cDNA。使用RNase H-的突变体反转录酶时，RNA仍然存在。RNase H可以去除与cDNA结合的RNA。但另一方面，有些人认为95°C的变性步骤会造成RNA引物与DNA脱离，因而RNase H处理完全没有必要。因此这一步骤是可选的。在克隆较大的片段时，RNase H处理的做法较为有益。
这很大程度上依赖于样品的质量。mRNA占总RNA的1–5%。根据所用的引物和酶，反转录反应可以将>70%的RNA转换成cDNA。
对于降解的RNA、含有严重的二级结构的RNA、无polyA尾的RNA或原核RNA，随机引物是最佳选择。随机引物仅推荐用于两步法RT-PCR，而且通常可以获得最高的得率，尽管cDNA可能不为全长cDNA。如希望通过两步法RT-PCR得到全长cDNA时，最好使用Oligo(dT)引物。这一反应受到二级结构和RNA质量影响。基因特异性引物应用于特异性基因的扩增反应，主要用于一步法RT-PCR反应。
不能再用了，必须更换DTT。
这些酶均含有RNase H结构域，但是这些RNase H结构域都经过突变。在RNase H活性检测分析之中，我们未检测到任何RNase H活性。
尽管实际体积取决于用于第一链合成反应的RNA起始量和靶标基因的丰度，但我们建议使用10%的第一链cDNA反应产物进行PCR反应。
If amplification products are generated in the control tube/well that contains no reverse transcriptase (i.e., the no-RT control), it may be necessary to eliminate residual genomic DNA from the RNA sample. Use the following protocol to remove genomic DNA from the total RNA preparation.Random primers are the best choice for degraded RNA, RNA with heavy secondary structure, non-polyadenylated RNA, or prokaryotic RNA. It is recommended only for two-step RT-PCR, and typically gives the highest yields, although the cDNA may not necessarily be full length. Oligo(dT) primers are good to use when trying to recover full-length cDNA from 2-step RT-PCR. The reaction is influenced by secondary structure and RNA quality. Gene specific primers should be used for very specific, mainly one-step RT-PCR reactions.
Random primers are the best choice for degraded RNA, RNA with heavy secondary structure, non-polyadenylated RNA, or prokaryotic RNA. It is recommended only for two-step RT-PCR, and typically gives the highest yields, although the cDNA may not necessarily be full length. Oligo(dT) primers are good to use when trying to recover full-length cDNA from 2-step RT-PCR. The reaction is influenced by secondary structure and RNA quality. Gene specific primers should be used for very specific, mainly one-step RT-PCR reactions.
Add the following to an autoclaved 0.5 mL microcentrifuge tube on ice:
1.Total RNA, ideally, less than or equal to 1 µg. (See Note 1 below.)
2.1.0 µL of 10X DNase buffer (200 mM Tris, pH 8.3, 500 mM KCl, 20 mM MgCl2).
3.0.1 U-3.0 U of DNase I (RNase-free, Cat. No. 18047019) or 1.0 U Dnase I, Amplification Grade (Cat. No. 18068015. (See Note 2 below.)
4.Bring volume up to 10 µL with DEPC-treated water.
5.Incubate at room temperature for 15 min. (See Note 3 below.)
6.Terminate the reaction by adding 1 µL 25 mM EDTA and heat 10 min at 65 degrees C. (See Note 4 below.)
7.Place on ice for 1 minute.
8.Collect by brief centrifugation. This mixture can be used directly for reverse transcription.
Please note the following:
1.To work with higher quantities of RNA, scale up the entire reaction linearly. Do not exceed 2 µg RNA in the 10 µL reaction. More RNA will increase the viscosity of the solution and prevent the DNAse I from diffusing and finding the DNA.
2.DNAse I, Amplification Grade has been extensively purified to remove trace ribonuclease activities commonly associated with other "RNAse-free" enzyme preparations and does not require the addition of placental RNAse inhibitor.
3.It is important not to exceed the 15 minute incubation time or the room temperature incubation. Higher temperatures and longer times could lead to Mg2+-dependent hydrolysis of the RNA.
4.This procedure requires careful pipetting of all solutions so that the concentration of divalent metal cation (Mg2+) is controlled.
5.Because the DNAse I must be heated to 65 degrees C to inactivate the enzyme, the concentration of free divalent metal ions must be low enough (less than 1 mM) after addition of the EDTA to prevent chemical hydrolysis of the RNA. See references below.
After the addition of EDTA, there is an approximately 1:1 molar ratio of Mg2+ :EDTA. EDTA chelates Mg2+ molecules on a 1:1 molar basis. Therefore, this RNA can be directly used in a reverse transcription reaction. First-strand reverse transcription buffers typically result in a final concentration of 2.5 mM Mg2+. If the reverse transcription buffer does not contain MgCl2, add it to the reaction at a final concentration of 2.5 mM. This results in a net final concentration of approximately 2.25 to 2.5 mM MgCl2.
References on RNA hydrolysis:
Molekulyarnaya Biologiya (1987) 21:1235-1241.
References on the mechanism of hydrolysis by other cations:
Eichorn GL and Butzov JY (1965) Biopolymers 3:79.
Butzov JY and Eichorn GL (1965) Biopolymers 3:95.
Farkas WR (1968) Biochim Biophys Acta 155:401.
The authors of the first paper express the opinion that the mechanism of the nonspecific hydrolysis by cations which proceeds through 2',3' cyclic phosphate formation is similar to that of specific hydrolysis such as RNA splicing.
The amount of RNA template for a cDNA synthesis is highly flexible and depends upon the amount of sample available and an individual's need. In general, 1 µg total RNA is used in a typical 20-µL RT reaction.
Find additional tips, troubleshooting help, and resources within ourReverse Transcription and RACE Support Center.
Some feel that the RNA in the RNA:DNA duplex after reverse transcription will inhibit PCR primers from annealing and amplifying the cDNA. The RNA is still present when using RNase H-mutant RTs. RNase H frees the cDNA from the RNA. On the other hand, some feel that the 95 degrees C denaturing step will cause the RNA primers to fall off the DNA and therefore RNase H treatment is not necessary. Therefore, this step is optional. For cloning of larger fragments, RNase H treatment can be beneficial.
This depends highly on the quality of the sample. mRNA itself makes up 1-5% of total RNA. Depending on the primer and enzyme used, reverse transcription can covert >70% of that into cDNA.
Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.
Random primers are the best choice for degraded RNA, RNA with heavy secondary structure, non-polyadenylated RNA, or prokaryotic RNA. It is recommended only for two-step RT-PCR, and typically gives the highest yields, although the cDNA may not necessarily be full length. Oligo(dT) primers are good to use when trying to recover full-length cDNA from 2-step RT-PCR. The reaction is influenced by secondary structure and RNA quality. Gene specific primers should be used for very specific, mainly one-step RT-PCR reactions.
Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.
No, the DTT will need to be replaced.
These enzymes contain the domains of RNase H, but they have been mutated. In RNase H activity detection assays, we are not able to detect any RNase H activity.
While the volume is dependent on the starting amount of RNA used for the first-strand synthesis and the abundance of the target gene, we'd recommend starting with 10% of the first-strand reaction for your PCR reaction.
We have not tested incorporation of biotin labeled nucleotides directly. Biotin should work as long as it is not substituted directly for its nucleotide replacement. Usually the biotin will be attached to one nucleotide such as TTP. We suggest that you try a reaction with a 1:1, 1:4, 1:10, and 1:50 replacement of the TTP in the reaction with biotin-labeled TTP. Most likely the 1:4 or 1:10 will work well. Having an excess of normal nucleotide around should space out the incorporation of biotin. This suggestion is speculative and has not been tested experimentally.
The optimal temperature for SuperScript III RT is 50 degrees C, and can be used up to 55 degrees C. For some qRT-PCR reactions where gene-specific primers are used, you can do the RT reaction at 60 degrees C. The optimal temperature for SuperScript II RT is 42 degrees C, and can be used up to 50 degrees C. Optimal temperature for MMLV is 42 degrees C. ThermoScript RT shows optimal activity at 60 degrees C, and can be used at temperatures as high as 70 degrees C (for amplicons expected to be 1 kb or less). For PCR products expected to be greater than 1 kb, a maximum first strand synthesis temperature of 60-65 degrees C is suggested. Be sure your first-strand primer anneals at the high temperature, especially when gene-specific primers are used for high-temperature stable reverse transcriptases. We recommend oligo (dT)20 for cDNA synthesis when using an oligo (dT) primer for first-strand synthesis with these enzymes.
Include a control reaction where the RNA has not been incubated with reverse transcriptase to test for specificity. If this RNA gives a PCR product, it is most likely generated from genomic DNA contamination. Alternatively, a primer set spanning two different exons can be designed such that the PCR product from the cDNA would be of a different size compared to a product generated from genomic DNA. Primers may also be designed to span an exon/exon junctions. These primers are not likely to amplify from genomic DNA templates. For DNase treatment of RNA, we recommend using Amplification-Grade DNase I, Cat. No. 18068-015, or an equivalent product.
It is not always necessary to digest the first-strand cDNA with RNase H. For many primer-template combinations, PCR products are seen without the RNase H treatment. Since SuperScript II and III RT lack RNase H and ThermoScript RT essentially is RNase H minus, the un-nicked RNA/cDNA hybrids may not denature well during the initial denaturation steps in PCR, leading to decreased sensitivity of the PCR reaction. These cDNA templates may require RNase H digestion. If a PCR product is not obtained when an RNase H step is not included after cDNA synthesis, always repeat the PCR after an RNase H treatment.
Yes, you can use a DNA-RNA hybrid as a template for M-MLV Reverse Transcriptase.
We have not tested this for SuperScript reverse transcriptases, so we cannot guarantee it would also work with those products.
This article can be used as a reference for additional information.
Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.
SuperScript II Reverse Transcriptase and SuperScript First-Strand Synthesis System for RT-PCR are shipped on wet ice. Our packaging and gel ice are designed to last approximately 48 hours before rising above 8 degrees C. Overnight incubation under these shipping conditions does not affect the SuperScript II Reverse Transcriptase enzyme's activity, performance, or applicable expiry date.
Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.