Hi Fellow EXPers!
Sorry it has been a while since my last post, it’s been a
crazy few weeks since my time ended in the lab. (college visits, summer work, hopefully I'm not alone here......) I officially finished my time in the lab the first week in August. The last 2.5 weeks in my lab were great, frustrating
at times, but I ended up getting results for my project, which is so awesome to
think about since I was only there for 6.5 weeks!
Lucky for me, these last two weeks were really the only time
that I experienced some frustration in my project. Since I had already extracted the DNA from
all of my 18hpf and 24hpf samples, the only step left was to run a good PCR so
all the bands were visible on the gel so that they could be cut and sent to be
sequenced, with the ultimate goal of getting clean sequences back to genotype
the fish. Seems simple, right? Yeah that's what I thought, but Not
exactly. From the start there are a few
things about this process that make it not exactly a piece of cake- 1. Because
I started with single embryos that weren’t that developed to start with, there
is limited DNA that I could extract in the first place which means that the PCR
could easily be faint on the gel and hard to amplify, and 2. The entire process of running the PCR, making a
gel, then running the samples, cut the bands, and preparing the sequencing
reaction takes much more time that you think, so having to start from the
beginning of the 2 hour PCR reaction every time is a bit of a pain, but
nonetheless is very doable.
It seemed that just about every time I ran the process,
something went wrong- the bands were too faint to cut, the samples were some how
contaminated, or worst of all I would get through the whole process, send the purified PCR off to
be sequenced and have the sequences stop 150 base pairs before the
mutation. Between the last two weeks, I
probably ended up running that whole process about 5-6 times (which is a lot, trust
me). The whole time, Patrizia and I continued to
troubleshoot and figure out why we couldn't get clean sequences; adding more eth. brom. to stain the gel, running the gel longer, and even
changing the sequencing core to Penn and not CHOP, and still nothing, for any of
the samples. We had just about tried
everything, when Patrizia had one last idea- try centrifuging for longer during
the purification to make sure to elute the DNA. So I ran the PCR one more time,
cut the bands, and centrifuged for double the amount of time to make sure we
had all the DNA we could possibly extract and sent the PCR off to CHOP (NAPCORE) for sequencing. Both of us were so anxious about getting the sequences
back, but when we opened the files, almost all the sequences worked for both
time points, which meant that we could genotype the fish!!! It was really so
exciting to finally know that I had a result and that I was able to help
Patrizia confirm what she thought for her paper!! After looking at the pictures I took before and comparing the different straining of the 3 genotypes, we concluded that there was really no difference in the expression of the
transcription factor, lmo2, in the mutant, heterozygous, or the wild type,
which means that the mutation must affect something else further downstream. Patrizia then helped me gather some pictures
and graphs for my poster, which was so nice and extremely helpful!
Aside from my main project with lmo2, while we were waiting for the sequencing results, I
started another project. My second
project involved going into the fish room, which so cool in itself and required me to cross an mll mutant fish with the new
transgenic line to look at the expression of the hematopoietic stem cells. On Wednesday night, Patrizia, Abby, and I all
went to the fish room, where I learned how to prepare the breeding tanks. Then, the next morning, I came in early to
learn how to heat shock the fish (we heat shock them in order to make
transgenic line expression MLL-GAS7, which has a heat shock promotor upstream
from the gene). I also learned how to
sort the fish after the heat shock, based on the presence of a green heart (how
we know that the fish are expressing MLL-GAS7).
Early the next week I helped Patrizia start the WISH, and although I didn’t
get to finish it, she said that she would finish it and send me the
results so that I could include them in my poster.
Overall, my experience in the Felix Lab this summer
was probably one of the best experiences I’ve ever had. I learned so much in such a short amount of
time and everyone in the lab was so welcoming and always there to help or
answer any question whenever I needed.
I thought that my project was great, it was just the right balance
between giving me freedom and feeling like I was doing something on my own, but
at the same time, I never felt overwhelmed with not knowing where on earth to
start or what to do next. My post doc was a fabulous mentor and never really made it feel like I was working. Six weeks goes by amazing fast, especially when the days don't even feel like work. I don’t think
I could have asked for a better lab team or environment to spend 2 months of my
summer in!
I hope that everyone had a great time in their labs! Can't to hear about everyone's summers in person in just a few short weeks!! See everyone soon! :)
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