Emily Eder HC70AL - Spring 2005 AT5g67300 Emily Eder HC70AL - Spring 2005

Arabidopsis Gene AT5g67300 Plant genotyping Sequencing and T-DNA insert RNA extraction and expression Using gel electrophoresis Using a genechip (microarray) Promoter Region

All Homozygous Mutants Genotypes All Homozygous Mutants

Genotypes cont.

AT5g67300 1216 Base Pairs in Length, Forward Orientation Codes for MYB Family Transcription Factor

Using Gel Electrophoresis to Determine RNA Expression

Using a Genechip (Microarray) to Determine RNA Expression

Promoter Region Why do we need to clone the promoter region of our gene? More accurate gene expression Why do we need to verify the sequence of the promoter region? Ensure that DNA sequence was not mutated

Promoter Sequence via Finch TV

Alignment Blast How can we verify that our sequence was not mutated? By blasting the two sequences together Verify mutations for both SP6 and T7

Using SP6 Primer No mutations

Using T7 Primer 1 mutation

Arabidopsis Gene AT6g67300 All Homozygous Mutant Plants One T-DNA insert with forward orientation RNA expression much higher in the leaf than the seed/silique Promoter region successfully cloned