Download What are the methods and approaches used to identify and

What Are the Methods and
Approaches Used to Identify
and Study Arabidopsis Seed
Knock-Out Mutations?
Daisy Robinton
Matt Emmer
Jason Chai
Isolation of DNA From Plant
Determining DNA Quality
How do we Know if There is an
Insert?
• Genotyping provides evidence for a T-DNA insert
• Genotyping is done by PCR amplification of the DNA
sequence of our gene with our gene-specific primers
along with a primer for the T-DNA insert
• We know of two ways to genotype
1. Multiplex Reaction
2. Separating Primers
How do we find the Insert via
Genotyping?
LB
FW
agggtcttctcgatgtagatatgcggaagat
RV
Multiplex Reaction
Wild Type
Wild Type
Heterozygous
Separating Primers
Sequencing Steps
–
–
–
•
•
•
•
•
•
Cut mutant band from gel
Sequence band
Set up sequencing reaction
Submit to sequencing center
Use Phred and Finch TV to analyze file
Determine T-DNA orientation
BLAST
Align 2 Sequences
Compare to expected results
Direction of Read
The Dideoxy Sequencing Process
Interpretation Sequencing Results
FINCH TV
Components of Performing a BLAST
The Bioinformatics Process
How to Determine a Mutant
Phenotype?
Phenotyping Plants:
- Observe phenotypical
differences between
wildtype and
homozygous mutants
- Study seeds with
Microscope
- Observe
embryological
differences by utilizing
the Nomarski
microscope