| | | | Product | | Tech Service: Amplification & Primers
RT-PCR, cDNA, and RACE Systems
| | | | Date Created | | | 06/10/1999 04:31 PM | | | | Date Updated | | | 09/12/2002 03:50 PM | | | |
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 E-mail Answer | | | | How should I prime my cDNA? | | | | Question | | | For first strand synthesis of cDNA, is it better to prime with oligo(dT), random hexamers, gene specific primer (GSP) or combination of these primers? | | | | Answer | | | It depends on your experimental goals. Oligo(dT) is used for many systems and is key for full-length cDNA. Random hexamers give a series of short first-strand products spanning the entire mRNA. Use of random hexamers may be helpful if your PCR fragment is at the 5´ end of a large mRNA. To ensure representation of the 3´ end, oligo(dT) can be added along with random hexamers. Gene specific primers (GSP) for cDNA synthesis may also be used and are required in a few applications such as 5´ RACE. In instances of 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 the different options of oligo dT, random primers and/or GSP for priming first strand synthesis may find a solution.
References:
FOCUS 12 p 47 available from Invitrogen Technical Services
FOCUS 14 p 91 available from Invitrogen Technical Services
Frohman,M.A., Dush,M.K., Martin, G.R. (1988) <i>Proc. Nat. Acad. Sci</i> USA 85, 8998
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