predicted Protocols

Category: Yeast Protocols: Yeast Protein Protocols: Yeast Protein Protein Interactions Protocols

This protocol describes the first step in constructing an array: amplification of the predicted ORFs that are to be included in the array. Gene-specific primers containing vector-specific flanking sequences that facilitate recombinational cloning are used to amplify each ORF. A secondary amplification can be used to extend the length of the homologous vector sequence flanking the ORF. - [Read Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: Amplification of ORFs]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Labeling Protocols

Hybridization is carried out in conventional aqueous solvents at a temperature well below the predicted melting temperature.  Nonspecific hybrids are then removed by washing at high stringency in buffers containing quaternary salts.  Tetramethylammonium chloride (TMACl) is used with probes that are 14-50 nucleotides in length, whereas tetraethylammonium chloride (TEACl) is used with longer oligonucleotides. - [Read Hybridization of Oligonucleotide Probes in Aqueous Solutions Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

A powerful way to identify a mutation in the gene of interest and to test mutant plants for phenotypes that are predicted to result from loss of function of that gene is by PCR screening. Pools of insertion lines are screened using one primer corresponding to the gene of interest and one primer corresponding to the end of the insertion element. The synthesis of a product indicates the presence of an insertion in the gene of interest. - [Read Screening DNA Pools for T-DNA Insertions in Arabidopsis Genes Protocol]