My first day without Julie.
It was definitely a little weird being there without her or knowing that
she wasn’t coming in right after me. But
even before I got to the lab my dad brought me in today instead of my mom and
when we were about five minutes away something happened with the oil in the car
and it keep beeping so I assumed it was pretty serious. We got to a parking lot we could stop in to
check out what happened. My dad looked
at the oil and he said he seemed as if there were none in there but my mom just
put a couple quarts in yesterday.
Luckily, we had some oil in the car so my dad filled it and hopefully
it’ll still be ok. When I finally got
into the lab, I talked about the crazy final yesterday in the Women’s World Cup
with one of the grad students. When we
finished talking, I went to go start the washes on my blot but unfortunately it
wasn’t in any primary. The last time Julie
or I were in the lab was Thursday and we couldn’t let the blot sit in the
primary for four days so this morning I got it out from the fridge and realized
I had to put primary on. I was stuck
because I wasn’t sure if I had to let it sit overnight and therefore would have
nothing to do for the day or if there was something else I could do. Luckily, Julie is still available to ask
questions and she said I could let it sit on the rocker for an hour and a half
at room temperature. During my waiting
time, I hung around with Eddie and helped him with anything he needed. I started the washes for the blot and in the
waiting time read my book as well as helped anyone who needed it. I made up the concentration for the secondary
when there was a little time left on the last wash. At first I couldn’t find my BSA in the fridge
and I was getting worried that we had used it up on one of the last washes but
right before I was going to ask someone, I spotted my name labeled on one of
the tubes. I grabbed that and took the
antibody and made up the solution. While
that sat for an hour I offered help and one of the newer grad students was
asking me about running a gel and setting up the transfer. That made me feel good because I knew what I
was doing and was able to help someone else. Once I finished helping him, I ate
my lunch and relaxed. When the hour was
up I began the washes once again and helped out during the breaks. I was finally done with all the washes and
could head down to the dark room. This
time I had really good luck and ended up with some nice films and when I came
up I scanned them in and analyzed them.
While I was doing this I was so happy because before Julie and I only
got one or two blots to work well enough to scan in and the fact that I got this
one to work while I was on my own was exciting.
That was the last test I’ll run with that specific blot so I put it in
TBSt and placed it in the fridge. I then
put the other blot we are working with in the proper primary so I could start
with the washes right away on Tuesday.
Tuesday began well.
The drive in was nice and when I got to the lab there were a few people
here before me. I asked Cagla about Fast
Green analysis and the timing for all the incubations and washes. I began that on the blot I was done with and
put in the fridge yesterday. Hopefully
I’ll be able to see all the bands on the blot.
I put on the destaining solution for a couple rinses and then let that
air dry. While I waited, I went in with
Kelly to look at the slides Julie and I had prepared last week. She checked them out to see what could
possibly have gone wrong during the transfection and the staining. I had to find the procedure we used and
determine what the secondary antibody was.
Luckily, I figured that out with Julie’s help, thank goodness for cell
phones and text messaging. I started the
washes for the other blot that I put the primary on yesterday and then put the
secondary on. During one of the breaks,
Cagla walked by with a tray of tubes and Shelly was right behind her and they
headed right for the tissue culture room with the hoods, as they were walking
Shelly asked all of us in the lab if we wanted to watch Cagla collect
monocytes. In the tubes was blood she
had collected from the dean of the dental school that she had centrifuged. She explained to all of us the specific layer
she wanted to collect and proceeded to do so.
With these monocytes, they will become macrophages in a matter of days
and then she will infect them with HIV for testing. It was pretty cool seeing the very beginning
of this process because then you know where the cells are coming from instead
of just having them right there and available to you. I ate lunch and one woman from another lab
came over and sat at the table and then Cagla sat with us as well, so we were
all talking and that was nice. I continued with the washes and went down to the
dark room. Unfortunately, nothing showed
up on the film, absolutely nothing. So I
went up and did another wash in TBSt then TBS.
This time I used the strong substrate and attempted to develop another
film. At first I didn’t think it was
that great because there wasn’t much on the film but I went upstairs and Cagla
asked how I was doing and she said that it was actually good data and that the
one band was E2F1 and it showed what we wanted it to. I put the next primary on and set that in the
cold room to sit overnight. I came back
in and then scanned the film in. All
went well with that and I’m set for tomorrow.
Wednesday started a little different. Today instead of just continuing with the
blot and the washes and everything, I ran a gel. I had run two gels previously but Julie had
always gotten everything ready for me and I just had to do the actual loading
so I didn’t know where a lot of the materials were. Luckily, Shelly, Eddie, and Anna were very
helpful and showed me where everything was and helped me figure out the right
amounts to load given how much protein was still left. I am very thankful for them. Then I went back to working on the blot I put
in primary yesterday. I had to go in and
get everything ready for the transfer which was difficult to time with the
washes on the blot. I was finally able
to figure everything out and put it together after a couple tries. Unfortunately, while I was doing this Cagla
asked if I used the microscope yesterday and I did with Kelly but I left it
on. I was supposed to turn it off in 45
minutes but I was so busy looking for the information Kelly wanted that I completely
forgot and it sat overnight. I feel
terrible because the bulbs are expensive and I left it on when it was
unnecessary. Anyway, my gel transferred
well onto the membrane and then I blocked it so that when I put the antibodies
on they will bind to the specific protein.
I continued with the secondary and the washes for the other blot. When the newer membrane was finished with the
blocking I put that on the next primary which is Synaptophysin. The other membrane finished with the washes
and I got everything ready to go down to the dark room. Unfortunately, when I developed it the first
one showed bands but there was a lot of background so I tried a couple more
times but no matter if I went for a shorter time or longer it barely showed
up. I went back upstairs and Eddie told
me to do two washes in TBS and then try again.
That time I only got one that was decent and even then it was
blurry. I’ll try again once I probe for
the other proteins. I decided to put it
in the next primary which is Axin2.
Cagla checked them again and agreed with my plan. Then I made more 5% BSA + TBSt because I use
that every time I make a secondary and then I used it to block so I was running
low. While I was waiting for the BSA to
dissolve I was sitting with my laptop and one of the grad students, Brigid, was
in today, she usually isn’t here because she is working in two labs and the
other is more of her focus. We were
talking about a bunch of different things and it was very enjoyable to relax
and chat. I finished out the day with a
little light reading.
Thursday was a little more challenging. I started the day with grabbing both blots
from the cold room and taking them out of the packaging and putting them in the
containers to wash. Just that first step
was more challenging than normal because I had to keep track of which blot had
which antibody and put them in the correct tubes. Luckily, the washes are the same so it’s not
that difficult but when I get to the secondary I have to be careful. I planned out what I’ll be doing for the rest
of the time I’m here so I can be organized and know what’s coming. I went and put the secondary on and got them
correct so that was the hardest part. I
thought about how I’m going to place the blots when I go down to the dark room
later. During my waiting time I followed
Eddie around and watched what he did. We
made more media but then he had a meeting to go to and my blots were finished
washing so I had to get ready and go down to the dark room. I warmed up his media by putting it in the water
bath and then put the blots side by side.
Since one had two ladders and the other had one it was easy to
differentiate between them but I still brought a Sharpie down with me to write
on the films as soon as they were developed.
The one ended up working out well and the bands were dark and that’s all
that was on there so hopefully that’ll scan in well and the other had a lot of
background. The bands looked good but
there wasn’t enough contrast so Cagla said to wash it overnight and put the
other in the fridge because I won’t be working with it for a while. Towards the end of the day I was following
Eddie around again and watched him feed his cells and then split another set of
cells. Cagla showed me the macrophages
that developed from the monocytes she collected yesterday. Then we all headed over to lab meeting.
Friday was definitely more stressful. I had to harvest protein from the cells Julie
plated last Tuesday and although I watched Eddie harvest protein and I myself
have harvested cells, it was still a little nerve-racking. But before I even started, I got here before
everyone else and usually Cagla is here early but she’s away and wasn’t here to
open the doors but luckily one of the newer dental students in the lab knew to
ask David, an administrator for the department.
He unlocked all the rooms we needed which was extremely helpful because otherwise
I wouldn’t have known what to do. When I
was finally able to start the procedure for harvesting, I used the directions
Julie had made up for me. They were very
detailed and I followed them to a T. I
also had Anna here to ask questions and she was also extremely helpful. But while I was harvesting the protein I was
also attempting to do a western. I had
let the one sit overnight washing in TBS and the day before Cagla said to wash
even more times the next day. So instead
of three washes I did five. I was
running back and forth between the rooms because in one I was using the
aspirator for the harvesting and in the other I had the western rocking. Finally, I got the cells into the tubes they
needed to be in and they had to sit on ice for 15 minutes and the western was
washing so I finally got a little bit of a break. I put the tubes in the centrifuge to spin
down all the cell debris to isolate the protein. Once those were done I collected the
supernatant and put it in another set of tubes.
I had to then quantify the protein by doing a Bradford assay which I’ve
done before with Julie. Unfortunately,
there wasn’t as much protein as I would’ve liked and I’m thinking maybe I didn’t
get all the PBS off when I aspirated it.
Anyway, I continued and made up the samples for when we do have to run a
gel with that protein and I put that in the -20º freezer and put the remaining protein in the -80º freezer. I was finally done with that part of my day
and all I had left was to focus on the blot.
That was done with the washes so I got it ready to go down to the dark
room and luckily it worked better than the day before. It still had a good amount of background but
it wasn’t as dark and you could see the band still so that made me happy. By time I finished everything my mom was
there to pick me up. All in all it was a
very busy day to finish off a productive week.
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