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Oligo(dT)20 Primer - FAQs

View additional product information for Oligo(dT)20 Primer - FAQs (18418020)

3 product FAQs found

Which components of the SuperScript III First Strand Synthesis System for RT-PCR are available for purchase separately?

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)

How does the Anchored Oligo(dT)20 Primer differ from standard oligo(dT) primers?

Anchored Oligo(dT) primers have 2 random bases at the 3' end of the stretch of Ts. The first random base is either an A, G, or C, while the second can be any of the 4 standard nucleotides, i.e., A, T, G or C. The use of anchored oligo(dT) primers results in increased cDNA synthesis yields.

For first-strand cDNA synthesis, is it better to use oligo(dT), random hexamers, gene-specific primer (GSP), or combination of these primers?

The choice of primer depends on your experimental goals. Oligo(dT) is recommended when using total RNA for cDNA synthesis. It is the key to full-length cDNA synthesis. Random hexamers give a series of short first-strand products spanning the entire mRNA. Use of random hexamers may be helpful if the PCR fragment is at the 5´ end of a large mRNA. To ensure full-length cDNA synthesis of large transcripts, oligo(dT) can be added along with random hexamers during first-strand synthesis. Gene-specific primers (GSP) for cDNA synthesis may also be used and are required in a few applications such as 5´ RACE and qRT-PCR. For GC-rich templates or templates rich in secondary structure, a GSP may not work as well as priming with oligo dT for first-strand synthesis. If an RT-PCR is problematic, trying different options of oligo dT, random primers and/or GSP for priming first-strand synthesis may resolve the issue. Oligo(dT)20 primer (Cat. No. 18418-020) is recommended for use with SuperScript III Reverse Transcriptase (Cat. no. 18080-044), ThermoScript Reverse Transcriptase (Cat. No. 12236-014), Thermo-X Reverse Trascriptase (Cat. No. 11150-025), and Cloned AMV Reverse Transcriptase (Cat. No. 12328-019).

Reference:
Frohman,M.A., Dush,M.K., Martin, G.R. (1988) Proc. Nat. Acad. Sci USA 85, 8998