Submitted to: American Society of Plant Physiologists Meeting
Publication Type: Proceedings
Publication Acceptance Date: June 1, 1999
Publication Date: N/A
We have constructed both cDNA and normalized cDNA libraries from soybean leaf and root. After the isolation of total RNA and mRNA from soybean leaf and root, first strand cDNA was synthesized using RNaseH- Superscript RT. Sal I and Not I linkers were incorporated during the cDNA synthesis for directional cloning into plasmid vectors. In order to construct a normalized cDNA library, biotinylated RNA was transcribed in vitro using plasmid DNA isolated from the total cDNA library. Excess amounts of biotinylated RNA were used to achieve a 500 fold Cot value through hybridization for the normalization of the cDNA library. For the EST study, individual colonies were archived from both the amplified cDNA library and the normalized cDNA library onto LB plates with ampicillin for plasmid DNA purification and onto FTA paper for long-term storage. More than 1,000 cDNA clones from the amplified cDNA library and normalized cDNA library were used to evaluate the EST sequences. Characterization and comparison of EST performed by (1) PCR for determination of cDNA length, (2) direct DNA sequencing for homology analysis, and (3) hybridization of tissue specific mRNA for the analysis of clones in the amplified cDNA library and normalized cDNA library will be discussed.