Step by Step SSCP

A very detailed protocol on SSCP by Travis Glenn. (in RTF word format)

This protocol is basically the same as any SSCP protocol you will find in the literature. The only major difference is that I recommend doing a two reaction PCR (unless you are screening more than 48 individuals, then you should optimize PCR conditions and amplify hot the first time around). The first reaction is a ¡°normal¡± cold PCR to amplify the intended products. Check that these reactions were successful by running out on a minigel. The second reaction is a hot PCR to label the products produced in the first PCR reaction. The hot product will be diluted in sequencing buffer, denatured, and loaded onto a native polyacrylamide gel. (Travis Glenn, Laboratory of Molecular Systematics, Smithsonian Institution)

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Last update 02-May-2007, Rating Excellent! of 1 votes.