So on Thursday afternoon of
last week our Primers finally came in. Unfortunately, since they came in a
small cardboard envelope they were easily misplaced and Adam and I had to run
all over the building that afternoon to try to find it. Turns out that a member
of our lab took it over to her bench because she thought it was for her. On
Friday Adam taught me how to rehydrate the primers since they are shipped in a
form without water. Earlier in the week we were supposed to go over to the old
building and grab some fresh A549 cells (adenocarcinomic human alveolar basal
epithelial cells) for DNA extraction to use as the template for PCR. We use
A549 cells because our lab has a ton of these cells since they provide good mediums
for which we can grow the influenza virus, plus the 45S gene we are looking for
is still present and functioning in those cells. It wasn’t until Friday,
however, that we were able to get those cells so instead of rushing the
extraction, Adam decided to dig through the freezer and find some old A549 DNA
and use that instead. Adam later left me in charge of picking the polymerase
and the settings for the thermal cycler. After a little research on my own (Adam
let me be in charge here), I decided to use the Q5 High-Fidelity Mastermix
because it contained basically every component necessary except the primers and
template while also being 100 times for accurate than Taq polymerase. So after
a lot of tedious benchwork, we were able to create 45S PCR amplicons.
We later ran our amplicons on a gel, and the amplicons
that turned up with bands were subsequently sent off for sequencing. In total,
14 amplicons (though some were of the same amplicon, just a different primer)
were sent off, and of those 14, none were sequenced successfully. A lot of the
results did have partial sequences though, so when we ran the sequences by
NCBI, in virtually all instances where there was a sequence to use, the
resulting region of genomic DNA was somewhere in the 45S gene. So while a lot
of red color might have turned up on the results page of the sequencing
website, we were able to confirm that the region of DNA sequenced was the same
region we wanted to sequence. To get full readings and better results, Adam and
I are looking into different parameters for PCR and perhaps a different method of
sequencing. This is very specific to our overarching DASH project. Our next
step is to use this sequence information and amplicons to design single-guideRNA
for CRISPR/CAS9 which should snip out the regions within the 18S, 5.8S, and 28S
Genes that are not variable leaving us with the ability to see how the variable
regions within the entire 45S gene on a whole function.
On a side note, Dr. Ghedin has been out of the lab on
vacation for the past 2 weeks so our entire lab decided to play hooky and go
hiking Bull Hill in Cold Spring, New York last Wednesday (7-13). Adam was the
one who orchestrated everything because he grew up in Rockland County. We all
met at Grand Central and took MetroNorth up to Cold Spring. From there, the 12
of us who went embarked on a 4 hour hike up and down this very steep mountain
that had gorgeous views of the Hudson Valley. It was nice to be able to finally
get to see members of my lab whom I only occasionally talk to anyway outside
the laboratory setting. Plus we were all able to bond about the tortures of
walking uphill under a bright sun in high 80 degree temperature. The views certainly
made the hike worthwhile though, and I did have the best shower and sleep of my
life when I got home that night.
(Results of our 45S PCR amplicons on a gel)
(Top of Bull Hill)
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