How to Achieve High Fidelity, Yield, and Full-Length Coverage with Transcriptor High Fidelity cDNA Synthesis Kit
Transcriptor High Fidelity cDNA Synthesis Kit: A Review
If you are looking for a reliable and efficient kit for first-strand cDNA synthesis, you might want to consider the Transcriptor High Fidelity cDNA Synthesis Kit from Roche. This kit is designed to produce high-quality cDNA with high accuracy, fidelity, yield, and full-length coverage. It also offers flexible priming options and RNase H activity for improved PCR sensitivity. In this article, we will review the features, advantages, and protocol of this kit, as well as provide some tips and troubleshooting advice. We will also show you where to find more information and download the PDF manual for this kit.
transcriptor high fidelity cdna synthesis kit pdf download
What is cDNA synthesis and why is it important?
cDNA synthesis is a process of reverse transcribing RNA into complementary DNA (cDNA) using a reverse transcriptase enzyme. cDNA synthesis is an essential step for many molecular biology applications, such as gene expression analysis, cloning, sequencing, mutagenesis, RNA interference, and CRISPR-Cas9 editing. By converting RNA into cDNA, you can preserve the information contained in RNA molecules, such as mRNA, miRNA, or lncRNA, and amplify it using PCR or other methods. cDNA synthesis also allows you to study RNA transcripts that are unstable, degraded, or present in low amounts.
What are the advantages of using Transcriptor High Fidelity cDNA Synthesis Kit?
High accuracy and fidelity
One of the main advantages of using this kit is that it provides high accuracy and fidelity in cDNA synthesis. This means that it reduces the errors and mutations that can occur during reverse transcription, such as base substitutions or frameshifts. These errors can affect the quality and reliability of your downstream applications, such as PCR or sequencing. The kit achieves high accuracy and fidelity by using a novel reverse transcriptase enzyme called Transcriptor Reverse Transcriptase, which has a proofreading activity that can correct misincorporated nucleotides. This enzyme also has a high thermostability that allows it to work at temperatures up to 65C, which can improve specificity and reduce secondary structures.
High yield and full-length cDNA
Another advantage of using this kit is that it provides high yield and full-length cDNA. This means that it produces high amounts of cDNA from a given amount of RNA, and that it can synthesize cDNA up to 14 kb in length. This is important for applications that require a large amount of cDNA or that target long transcripts, such as mRNA or lncRNA. The kit achieves high yield and full-length cDNA by using a high concentration of Transcriptor Reverse Transcriptase and an optimized reaction buffer that contains potassium phosphate, magnesium chloride, and dithiothreitol. These components enhance the enzyme activity, stability, and processivity, allowing it to synthesize cDNA efficiently and completely.
Flexible priming options
A third advantage of using this kit is that it offers flexible priming options for cDNA synthesis. This means that you can choose different primers depending on your RNA template and your downstream application. The kit provides two types of primers: random hexamer primers and anchored-oligo(dT)18 primers. Random hexamer primers are short oligonucleotides that can bind to any RNA sequence, and are suitable for synthesizing cDNA from any type of RNA, such as mRNA, miRNA, or lncRNA. Anchored-oligo(dT)18 primers are longer oligonucleotides that can bind to the poly(A) tail of mRNA, and are suitable for synthesizing cDNA from mRNA only. You can also use your own specific primers, such as gene-specific primers or universal primers, if you want to target a specific RNA sequence or region.
RNase H activity for improved PCR sensitivity
A fourth advantage of using this kit is that it has RNase H activity for improved PCR sensitivity. This means that it can degrade the RNA in RNA:DNA hybrids after cDNA synthesis, which can increase the accessibility and availability of cDNA for PCR primers. This can improve the specificity and efficiency of your PCR reaction, and reduce the background noise and artifacts. The kit has RNase H activity because Transcriptor Reverse Transcriptase has an endogenous RNase H activity that can digest RNA in RNA:DNA hybrids. You can also add an external RNase H enzyme, such as Protector RNase Inhibitor, which is provided with the kit, to enhance the RNase H activity.
How to use Transcriptor High Fidelity cDNA Synthesis Kit?
Kit components and storage conditions
The kit contains all the reagents required for first-strand cDNA synthesis reactions, including Transcriptor Reverse Transcriptase, RT Reaction Buffer, Protector RNase Inhibitor, Deoxynucleotide Mix, random hexamer primers, anchored-oligo(dT)18 primers, and water. The kit also contains control reagents for quality control purposes, such as control RNA and control primer mix. The kit comes in different sizes depending on the number of reactions you need: 50 reactions (Cat. No. 05081955001), 100 reactions (Cat. No. 05091284001), or 200 reactions (Cat. No. 05081963001). You can store the kit at -15C to -25C for up to 12 months.
Protocol overview
The protocol for using this kit is simple and straightforward. You just need to follow these steps:
Prepare your RNA template by purifying it from your sample using an appropriate method, such as High Pure RNA Tissue Kit (Cat. No. 12033674001). You can use any type of RNA template, such as total RNA, mRNA, miRNA, or lncRNA, depending on your application. You can also use synthetic RNA or in vitro transcribed RNA as templates.
Determine the amount and quality of your RNA template by measuring its concentration and purity using a spectrophotometer or a fluorometer, and its integrity using a gel electrophoresis or a capillary electrophoresis system.
Select your primer system according to your RNA template and your downstream application. You can use random hexamer primers for any type of RNA template, anchored-oligo(dT)18 primers for mRNA template only, or specific primers for targeting a specific RNA sequence or region.
```html Incubate your cDNA synthesis reaction at the appropriate temperature and time according to your primer system and RNA template. The recommended incubation conditions are 55C for 10 minutes for random hexamer primers, 50C for 50 minutes for anchored-oligo(dT)18 primers, and 55C for 30 minutes for specific primers.
Terminate your cDNA synthesis reaction by heating it at 85C for 5 minutes to inactivate Transcriptor Reverse Transcriptase. You can also add RNase H enzyme to digest the RNA template if desired.
Store your cDNA product at -20C or use it directly for your downstream application, such as PCR or qPCR.
Tips and troubleshooting
Here are some tips and solutions for common problems when using this kit:
If you have low or no cDNA yield, check the quality and quantity of your RNA template, use fresh and nuclease-free reagents and labware, optimize your primer concentration and annealing temperature, increase your incubation time, or use a carrier nucleic acid to improve cDNA precipitation.
If you have low cDNA specificity or high background noise, check the purity and integrity of your RNA template, use specific primers instead of random hexamer primers, reduce your primer concentration and incubation temperature, or use a hot start PCR enzyme to prevent nonspecific amplification.
If you have low cDNA fidelity or high mutation rate, check the quality and stability of your reverse transcriptase enzyme, use fresh and nuclease-free reagents and labware, optimize your deoxynucleotide concentration and incubation temperature, or use a proofreading PCR enzyme to correct errors during amplification.
If you have low cDNA length or incomplete transcripts, check the quality and integrity of your RNA template, use anchored-oligo(dT)18 primers instead of random hexamer primers, increase your reverse transcriptase concentration and incubation time, or use a high processivity PCR enzyme to amplify long fragments.
Where to find more information and download the PDF manual?
If you want to find more information about this kit, such as product specifications, performance data, customer reviews, FAQs, citations, and related products, you can visit the product website at https://www.sigmaaldrich.com/US/en/product/roche/thificdnaro. You can also contact the customer service or technical support team if you have any questions or feedback about this kit.
If you want to download the PDF manual for this kit, which contains detailed instructions, protocols, safety information, storage conditions, quality control data, ordering information, and references, you can click on the link below:
Transcriptor High Fidelity cDNA Synthesis Kit Manual
Conclusion
In conclusion, the Transcriptor High Fidelity cDNA Synthesis Kit is a reliable and efficient kit for first-strand cDNA synthesis that offers high accuracy, fidelity, yield, and full-length coverage. It also provides flexible priming options and RNase H activity for improved PCR sensitivity. It is suitable for various molecular biology applications that require high-quality cDNA from different types of RNA templates. It is easy to use and has a simple protocol that can be completed in less than an hour. It also has a comprehensive PDF manual that contains all the information you need to use this kit successfully.
FAQs
Here are some frequently asked questions and answers about this kit:
What is the difference between Transcriptor High Fidelity cDNA Synthesis Kit and Transcriptor First Strand cDNA Synthesis Kit?The main difference is that Transcriptor High Fidelity cDNA Synthesis Kit uses Transcriptor Reverse Transcriptase, which has a proofreading activity that can correct misincorporated nucleotides during cDNA synthesis. This results in higher accuracy and fidelity than Transcriptor First Strand cDNA Synthesis Kit, which uses Transcriptor Reverse Transcriptase without proofreading activity.
How can I quantify and analyze my cDNA product?You can quantify your cDNA product by using a spectrophotometer or a fluorometer to measure its concentration and purity. You can also use a gel electrophoresis or a capillary electrophoresis system to check its size and integrity. You can analyze your cDNA product by using PCR or qPCR to amplify specific regions or genes of interest, or by using sequencing or microarray to profile the whole transcriptome.
How can I store and use my cDNA product?You can store your cDNA product at -20C for up to 12 months. You can use your cDNA product directly for your downstream application, such as PCR or qPCR, without further purification or dilution. You can also use your cDNA product for other applications, such as cloning, mutagenesis, RNA interference, or CRISPR-Cas9 editing, after appropriate purification and modification steps.
What are the limitations and precautions of using this kit?Some limitations and precautions of using this kit are: it is not suitable for cDNA synthesis from RNA templates that contain strong secondary structures, such as GC-rich regions or hairpin loops; it is not compatible with RNase inhibitors that contain guanidine salts, such as RNaseOUT; it may not work well with RNA templates that are highly degraded, contaminated, or present in low amounts; it requires careful handling and storage of the reagents and labware to prevent nuclease contamination and degradation.
What are some alternative kits for cDNA synthesis?Some alternative kits for cDNA synthesis are: SuperScript IV First-Strand Synthesis System, which uses an engineered MMLV reverse transcriptase that has high processivity, thermostability, and resistance to inhibitors; Maxima H Minus First Strand cDNA Synthesis Kit, which uses a recombinant M-MuLV reverse transcriptase that has reduced RNase H activity and increased thermal stability; Xscript I+DNA-Y cDNA Synthesis Kit, which uses a novel reverse transcriptase that has high fidelity, efficiency, and specificity.
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