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View additional product information for SuperScript™ III Reverse Transcriptase - FAQs (18080093, 18080044, 18080085)
37 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。
可以。我们单独出售 的M-MLV反转录反应缓冲液（货号18057018），可以用于M-MLV反转录酶、SuperScript II反转录酶和SuperScript III反转录酶。
不会。在加入EDTA后， Mg2+:EDTA的摩尔比约为1:1。EDTA会与Mg2+按照1:1的摩尔比例进行螯合。因此RNA可以直接用于反转录。第一链反转录缓冲液的Mg2+终浓度通常为2.5mM。 如果反转录反应缓冲液中不含MgCl2，则可向反应体系中添加终浓度为2.5 mM 的MgCl2 。因此会使MgCl2的净终浓度约为2.25-2.5 mM。
建议使用酶附带的缓冲液。之所以出现这些细微的差异，是因为不同的试剂盒开发时间不同，并且很可能由不同的研发团队开发。
不会，如果需要有TdT活性的酶，请使用SuperScript II反转录酶。
SuperScript VILO cDNA合成试剂盒含有SuperScript III反转录酶和辅助蛋白的混合物，其中后者可以提高反转录反应的效率，进而提高得率。由于存在辅助蛋白，SuperScript VILO中的反转录在42°C条件下活化。
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。
The following components are available as stand-alone items:
- Superscript III Reverse Transcriptase (Cat. Nos. 18080093, 18080044, 18080085)
- Oligo (dT)20 Primer (Cat. No. 18418020)
- Random hexamers (Cat. No. 48190011)
- 10 mM dNTP Mix (Cat. Nos. 18427013, 18427088)
- RNAseOUT Recombinant Ribonuclease Inhibitor (Cat. No. 10777019)
- E. coli RNAse H (Cat. Nos. 18021014, 18021071)
SuperScript III Reverse Transcriptase (Cat. Nos. 18080093, 18080044, 18080085) contains the stand-alone enzyme and a vial each of 5X first-strand buffer and 100 mM DTT.
SuperScript III First Strand Synthesis System for RT-PCR is a complete kit that provides the SuperScript III Reverse Transcriptase and all the other components required for synthesis of first-strand cDNA from total or poly(A)- RNA. It includes:
- Superscript III Reverse Transcriptase
- Oligo (dT)20 Primer
- Random hexamers
- 10X RT buffer
- 25 mM MgCl2
- 0.1 M DTT
- 10 mM dNTP Mix
- RNAseOUT Recombinant Ribonuclease Inhibitor
- E. coli RNAse H
- DEPC-treated water
- Total HeLa RNA control
- Sense control primer
- Anti-sense control primer
Note: The kit does not include the PCR amplification enzyme.
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.
Yes, we sell a M-MLV RT buffer (Cat. No. 18057018), which works with M-MLV RT, SuperScript II RT, and SuperScript III RT.
No. 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.
It is recommended to use the buffer that comes supplied with the enzyme. The reasons for the slight differences are that the kits were developed at different times, possibly by different R&D groups.
No, if TdT activity is required please use our SuperScript II RT.
The SuperScript VILO cDNA Synthesis Kit contains a mix of SuperScript III RT and helper proteins which help to increase the efficiency of the reverse transcription reaction and thus improve yield. The RT in the SuperScript
The SuperScript VILO cDNA Synthesis Kit (Cat. No. 11754050) contains a mix of SuperScript III RT and helper proteins which help to increase the efficiency of the reverse transcription reaction and thus improve yield. The RT in the SuperScript VILO kit is active at 42 degrees C due to the helper proteins.
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.
The purpose of the 25 degrees C incubation step when using random hexamers is to enhance binding to the template. Since the primers are short, a lower annealing temperature is required. This step can be omitted if you use a "cold-start" in the next step (i.e., ramp up gradually to 50 degrees C). If you use a hot-start (pre-heated to 50 degrees C), and the 25 degrees C step has been omitted, you will not get good first-strand synthesis because the temperature will be too high for efficient annealing.
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.
A low number of cDNA clones could result due to the reasons listed below:
(1) Phenol extractions were performed with phenol that was equilibrated with water that was not DEPC-treated.
(2) Poor first strand yield.
(3) No RNase H was added to second-strand reaction.
(4) Second-strand reaction was performed at a temperature greater than 16 degrees C.
(5) Dilution of first-strand reaction was made incorrectly: exact dilution is crucial, because the pH of the second-strand reaction differs from that of the first-strand reaction.
(6) The size fractionation column ran too quickly, the column was allowed to dry before fractions were loaded, or the column was not washed thoroughly to remove ethanol.
(7) Competent cells were of poor efficiency: verify efficiency of transformation with a control DNA.
(8) There was an insufficient amount of cDNA in the ligation mix.
(9) If cells were transformed by electroporation, there may have been too much salt in the electroporation mix, which kills cells.
Please see listed below a few reasons why you may be getting small first-strand cDNA products:
(1) Template was degraded by RNase contamination: maintain aseptic conditions.
(2) Secondary structure is present in the RNA:
-Heat the RNA to 70 degrees C for 10 min and quick-chill on ice to denature the RNA. Increase the temperature of the first strand reaction up to 50 degrees C. Denature the RNA by treatment with 20 mM methylmercuric hydroxide (see Krug et. al. (1987) Methods Enzymol 152:316).
-Try alternate reverse transcriptase such as SuperScript III polymerase and perform first-strand synthesis at higher temperature.
(3) Alkaline gel electrophoresis was performed incorrectly: use a 1% alkaline agarose gel.
(4) For random-primed first-strand cDNA, an excessive concentration of primers was used: use 50 ng random hexamers/1-5 µg total RNA.
(4) Incorrect fractions were taken at the column chromatography step.
The enzymes can be inactivated by adding a chelating agent such as EDTA. Alternatively, with the exception of ThermoScript RT and Thermo-X RT, the enzymes can be heat inactivated at 70 degrees C for 10 min.
ThermoScript RT should be heated to 85 degrees C for 5 min for complete inactivation.
For Thermo-X RT, if using an oligo(dT) primer, add EDTA to the reaction at a final concentration of 5 mM. Inactivate the reaction by heating at 90 degrees C for 5 min.
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.
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.
You can store the cDNA at -20 degrees C for up to 1 week. For long-term storage, we recommend storing the cDNA at -70 degrees C.