1. Searching for a Knockout Line for a Gene of Interest in Arabidopsis
GOAL: Identify a target plant line among 60,480 individual T-DNA transformed lines
Wisconsin Madison Facili ty
Screen for 1 out of 30 super pools (2,025 T-DNA lines per super pool)
Screen for 1 out of 9 DNA pools (225 T-DNA lines per DNA pool)
Screen for 1 out of 25 seed pools (9 T-DNA lines per seed pool)
Screen for 1 out of 9 seed lines f
or the target gene tagged
with T-DNA (1 T-DNA line per seed line)
Determine genotype of each plant
to find T-DNA target line by PCR
Heterozygote
(a/A)
Homozygote
(a/a)
The target gene may
be INVOLVED in
seed development No
Yes
The target gene may
Collect seeds for further
or may not involved
analysis in the next
in seed development
generation
(duplicate gene??)
Examine plants for mut
ant phenotype
Collect seeds for duplicat
e gene crosses