Journal: Methods in enzymology
Article Title: Methods for Genome-Wide Analysis of Gene Expression Changes in Polyploids
Figure Lengend Snippet: Amplified fragment length polymorphic complementary DNA (AFLP-cDNA) display analysis of gene expression. (A) A simple diagram of the AFLP-cDNA display procedures including messenger RNA (mRNA) isolation, cDNA synthesis, restriction digestion, ligation, preselective and selective amplification, and gel electrophoresis. The detailed protocols are described in the text. Only a few hypothetical fragments are shown to illustrate how Eco R I (GAATTC)- and Mse I (TTAA)–digested fragments are ligated and amplified during the analysis. (B) AFLP-cDNA display analysis using leaves of Arabidopsis thaliana diploid (At2), autotetraploid (At4), Arabidopsis arenosa (Aa), and generations 2–5 (S2-5) of new allotetraploids, and a natural Arabidopsis suecica ) are not shown because of space limitations.
Article Snippet: To digest cDNA, add 5 μ l of 5× reaction buffer, about 18 μ l of cDNA (~250 ng), 2 μ l of Eco R I/ Mse I (1.25 U/ μ l each) as supplied by the manufacturer (Invitrogen) to a final volume of 25 μ l. Mix gently, centrifuge briefly, and incubate the reaction at 37° for at least 2 h. The reaction is inactivated by heating at 70° for 15 min.
Techniques: Amplification, Expressing, Isolation, Ligation, Nucleic Acid Electrophoresis