Hazel Ruth Denney

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Hazel Ruth Denney

• Makah Potato Genomics Workshop
• June 22-July 3, 2003
• USDA/ARS National Science Foundation

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The hypothesis of this work shop is to find
where the Ozette potatoe originated from
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The Important people

Left to right Chuck brown Cassandra Smith Hazel
r denney Tia Beavert Neuee v. Ward David
culley Daniel Andrade
The Research bunch
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The first day on the job
On the first day we took various notes and
learned how to construct DNA models.
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Typical day in the class room learning the
fundamentals of dna extraction
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Lab
Getting familiar with the lab, learning the
protocol, starting the experiment.
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Plant DNA Extraction


 
   
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Protocol     Grind leaf tissue in a
pre-chilled mortar in liquid nitrogen to a fine
powder. 2.       Transfer 100mg powder into an
Eppendorf tube. Add 700µl of fresh 2X CTAB buffer
(and 2µl of ß-mercaptoethanol and vortex). Place
in 65C water bath for 45 min, inverting tubes
every 15 min. Cool samples to RT for 2
min. 3.       Add 700µl of chloroformisoamyl
alcohol (241) to each tube. Vortex briefly and
gently to avoid shearing DNA, then turn over
several times. 4.       Spin 5 min at 14,000 rpm
in a microcentrifuge. Remove aqueous top layer
and transfer to a new labeled Eppendorf tube. Be
careful to avoid carrying over interphase
material. Dispose chloroformisoamyl alcohol
waste properly in a labeled waste
container. 5.       Add 50µl of 10 CTAB (in 0.7
M NaCl), vortex gently, and mix
thoroughly. 6.       Repeat steps 3 and 4.
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7.       Add equal volume of cold (-20C)
isopropanol (400-500µl) to each tube. Turn over
several times and let tubes sit at 4C for 30 min
(15 min at -20C or 5 min at 70C). 8.      
Spin at 14,000 rpm for 20 min. Carefully pour off
supernatant so as not to lose the DNA pellet
turn over tubes to dry (1-2 min). 9.       Wash
pellet with 1ml 70 EtOH (at least 1-3 min) and
spin 30 min at 14,000 rpm. Carefully pour off
EtOH, wash pellet in 1ml 95 EtOH, spin 30 min at
14,000 rpm, and carefully pour off EtOH. Turn
tubes upside down and air-dry overnight or for
few minutes using a vaccum pump. 10.   Dissolve
DNA in 150µl T10E1 or distilled H2O per sample,
add 1-2µl 10mg/ml (DNAse-free)-RNAseA, vortex
briefly and gently, and spin briefly. Incubate at
37C for 1 hr. 11.   DNA concentration is
estimated in comparison with known concentration
of lambda DNA in 1 agarose gel. 12.   Store at
4C, or at -20C for long-term storage.
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Doing the experiments
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Hard AT WORK
Cassandra is loading the sequencing gel to
separate the DNA by size
Here we are naming the tubes
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The final result of the DNA extraction
Science does not always work out the way you want
to !!!! The first experiment was a flop.
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The reason the first trial run did not work is
because something went wrong with the PCR. We are
not really sure why but there was no PCR product.
We checked the DNA and the template was fine. So
that means there was something wrong with the
master mix or the primers.
What went wrong
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How we fixed our problem
To try and fix the problem we had to start
fresh. We had to make everything brand new. We
made a new master mix. Then we redid everything
but we were much more careful about our
procedures.
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Our second trial
On this trial we had to work long hours and
weekends
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Trying it all over again
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The Final result of the second trial
It was a success it worked out perfect
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The Nemotode Presentation
Presented by Hassan Mojahedi
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Nematode
Any of several worms of the phylum Nematode,
having unsegmented, cylindrical bodies, often
narrowing at each end, and including parasitic
forms such as the hookworm and pinworm. Also
called roundworm.
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Various pictures of Nematodes
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THE EFFECTS CERTAIN NEMOTODES HAVE ON POTATOES
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This is a Norkotah Russet Potato that has been
damaged by the Columbia Root Knot nematode
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Various types of Potatoes
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Conclusion
I would like to thank everybody that made it
possible for me to be a part of this experience.
A special thanks to Dr. Charles Brown, Kay
Brown, Dave Culley, Daniel Andrade, Neuee V.
Ward, Gary Giovane,All the people that shared
their presentations with us, and several involved
community members.