Sistema de síntesis de primera cadena SuperScript™ para RT-PCR

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.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.