segregating Ds insertion lines

C. Robb Flynn Robb.Flynn at asu.edu
Mon Feb 5 18:36:28 EST 2001


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I have some rudimentary questions on Arabidopsis Ds insertion mutagenesis
that I can't seem to find the answers to.&nbsp; The answers are probably
there, I just don't understand them.&nbsp; I have read both of Dr. Sundarasen's
papers, a number of different websites, and the <i>Arabidopsis protocols</i>
boo, yet I am still unclear on a few things.&nbsp; I would very much appreciate
anyone's time and answers to the following:
<p>1.&nbsp; In regards to the 931 characterized IMA lines described by
Sundarasen that are currently being distributed by NASC, are the seeds
being distributed from the F2 or F3 generation?
<p>2.&nbsp; Can you briefly describe the percent germination numbers expected
for a homozygote, or a heterozygote Ds insertion, at each the F2, F3, and
F4 stages if the seeds are grown on Kan/NAM?&nbsp; Is it possible to tell
the difference between the two based on germ. rates alone?
<p>Basically, I can't yet figure out if I have segregated the seeds enough
to verify that I have a homozygous knock-out phenotype.&nbsp; My germination
numbers are described below (if anyone has the time) but answers to the
above questions should help me figure out where I stand. I realize that
a Southern blot would give me an answer to my question, and I have PCR
data verifying at least one copy of the insertion in the right gene, but
I was hoping germination rates alone could tell me homo vs. hetero.
<p>Thanks,
<p>Robb Flynn
<p>robb.flynn at asu.edu
<br>&nbsp;
<p>Upon receiving a line (~50 seeds) from NASC, I started about 25 seeds
(some on Kan/NAM plates,&nbsp; some on dirt - I did not keep track of numbers
), however only 18 germinated and gave seedlings.&nbsp; All plants were
eventually transferred to dirt and selfed.&nbsp; Seed was collected from
each plant and kept separate (I know which was on dirt and which was on
media).&nbsp; About 200 seeds (1 batch) from each of these plants were
germinated on Kan/NAM plates.&nbsp; 9 of the 10 batches of seeds from plants
initially germinated on dirt were selected against and died (yellow, small,
aborted). The other batch had a&nbsp; ~66% germination rate.&nbsp; 5 of
the 7 batches of seed collected from plants initially germinated on Kan/NAM
showed a germination rate of ~66%, the other two batches had germination
rates of ~75%.&nbsp; Based on this information can you tell me if any of
my seed batches is homozygous for the transosable element/knockout?</html>

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