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From RNA to sequenced clones within three days: a complete protocol.

Detection of specific mRNA transcripts by the reverse transcription/polymerase chain reaction (RT/PCR) technique has become increasingly important. The technique is fast and has a very high resolution. Cloning of these PCR fragments into vectors is sometimes necessary for identification of alternative splicing products, for bacterial expression or for generation of a DNA probe. Here we present a complete protocol for RT/PCR, cloning and sequencing of PCR, cloning and sequencing of PCR products beginning with the total RNA and ending with the DNA sequence within three days. To illustrate the procedure as an example, a fragment of the human glyceraldehyde-3-phosphate dehydrogenase mRNA was amplified from total RNA, cloned and partially sequenced. The protocol has been optimized for small scale to facilitate handling and to reduce costs.