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Invitrogen™ SuperScript™ First-Strand Synthesis System for RT-PCR
Description
Includes
50μL Oligo(dT)12-18 (0.5μg/μL); 250μL Random hexamers (50ng/μL); 1mL 10X RT buffer; 500μL 25mM Magnesium Chloride; 250μL 0.1M DTT; 250μL 10mM dNTP mix; 50μL SuperScript II RT (50U/μL); 100μL RNaseOUT (40U/μL); 50μL E. coli RNase H (2U/μL); 1.2mL DEPC-treated water; 15μL Control RNA (50ng/μL); 20μL Control Primer A (10μM); 20μL Control Primer B (10μM)
The SuperScript First-Strand Synthesis System for RT-PCR is optimized for the synthesis of first-strand cDNA from purified poly(A)+ or total RNA. The system can be used with as little as 1 ng or as much as 5 μg of total RNA. After synthesis, the cDNA can be amplified with specific primers by PCR without intermediate organic extractions or ethanol precipitations. In conjunction with PCR, the system can be used to detect the presence of rare messages, to quantitate the amount of specific mRNA from small numbers of cells, or to clone specific cDNAs without constructing an entire cDNA library. The system is flexible, allowing the use of any PCR enzyme. Combine with AccuPrime Taq DNA Polymerase or Platinum Taq DNA Polymerase for higher specificity PCR or with AccuPrime Pfx DNA Polymerase for high-fidelity cloning applications.
SuperScript II RT
The first-strand cDNA synthesis reaction is catalyzed by SuperScript II Reverse Transcriptase (RT). This enzyme has been engineered to reduce the RNase H activity that degrades mRNA during the first-strand reaction, resulting in greater full-length cDNA synthesis and higher yields of first-strand cDNA than obtained with RNase H+ RTs. Because SuperScript II RT is not inhibited significantly by ribosomal and transfer RNA, it may be used effectively to synthesize first-strand cDNA from a total RNA preparation. The enzyme exhibits increased thermal stability and may be used at temperatures up to 50°C.
Using the SuperScript First-Strand Synthesis System for RT-PCR
This system has been optimized to synthesize first-strand cDNA from varying amounts of starting material. The SuperScript II RT concentration has been lowered and RNaseOUT Recombinant RNase Inhibitor has been added to the system as part of this optimization process. Additionally, reaction conditions have been modified to further increase the sensitivity of the system. Using the kit, you synthesize first-strand cDNA using either total RNA or poly(A)+ selected RNA primed with oligo(dT), random primers, or a gene-specific primer. Then perform PCR in a separate tube using primers specific for the gene of interest.
Order Info
Shipping Condition: Wet ice
Specifications
Specifications
| Content And Storage | • Oligo(dT)12-18, 50 μL (0.5 μg/μL) |
| Format | Kit |
| GC-Rich PCR Performance | High |
| Reaction Speed | 50 min. |
| Technique | Reverse Transcription |
| Optimal Reaction Temperature | 42°C |
| Primer | Random Primers, Oligo dT Primers |
| Quantity | 50 rxns |
| Reverse Transcriptase | SuperScript II |
| Shipping Condition | Wet Ice |
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Frequently Asked Questions (FAQs)
You can store your cDNA at 2-6 degrees C for up to 24 hours. For long-term storage, store the cDNA at -15 to -25 degrees C and add EDTA to a final concentration of 1 mM to prevent degradation.
Degenerate primers are a mixture of oligonucleotides that are all the same length but have more than one possible base at some of the positions. Degeneracy of the genetic code for the selected amino acid is generally the reason for needing/using degenerate primers for PCR. Degenerate primers are used in the following research scenarios:
(1) Locating the corresponding gene from the sequence of a protein that has been purified/isolated.
(2) Finding gene homologues across species. If the sequence of a particular gene is published across several species, there will be some universal homology and some degeneracy, so finding a homologue in a new species may require degenerate sequences for amplification.
(3) Locating related genes across species.
(4) Phylogenetic and evolutionary studies.
(5) Studying gene families.
Guidelines for designing degenerate primers
-Try to use primers that are a minimum of 20 bases.
-Selection of amino acids with minimal degeneracy is desired (amino acids requiring less than 4 or 6 codons). Serine, arginine, and leucine are the amino acids with the most wobbles.
-Consider codon bias for translation depending on the gene, organism, or virus!!!
-In addition to using degeneracy to compensate for the variance in the genetic code, it can also be used to substitute similar types of amino acids such as Glu--Asp and Arg--Lys.
-Degeneracy greater than 516-fold is not recommended. PCR has been performed using primers with higher degeneracy, but it is very inefficient. The degree of degeneracy depends on the number of degenerate positions and the number of bases used at these particular positions. E.g., ACC TGG CAA TTT CGG IGG CGT. Degree of degeneracy is 3 X 2 X 4 (3 possible bases at position 4, 2 possible bases at position 7, and 4 possible bases at position 16).
-Try to avoid degeneracy at the 3' end of the primer if possible. 6-9 base 5' extensions can be added. While not complementary to the template, these 5' extensions become incorporated into the amplified product at the second and all subsequent cycles. It increases the overall efficiency of PCR by increasing the stability of the duplex.
-Inosine-containing primers are also used because of the ability of this base to pair with all four bases. The drawback is that inosine (I) residues decrease the Tm of the primer.
-Avoid using degenerate primers to amplify large amplicons (>1,000 bp).
-Empirical testing of the designed primers is required. Consider a two step PCR when using degenerate primers, where the first 4 or so cycles are performed at a low annealing temperature and then linking it to a new program that steps up the annealing temperature 5-10 degrees C. Sometimes additional cycles are needed to generate sufficient product when performing degenerate PCR.
References
(1) Degenerate Primers in PCR, in PCR Protocols. A Guide to Methods and Applications. MA Innes, DH Gelfand, and JJ Sninsky eds (1990), chapter 5.
(2) PCR Protocols, Current Methods and Applications, in Methods in Molecular Biology. BA White ed (1993), chapter 31.
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.
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.
For Research Use Only. Not for use in diagnostic procedures.