Going into this week, I was a little bit terrified. My grad student, being away this week, had left me a list of tasks to complete and some protocols. I really did not want to have to send a text to her saying, "Please help. I messed up a lot!" (spoiler alert: I didn't have to) My first job on Monday was to rinse a primary antibody off of western blots and to then put on a secondary. I was in charge of four westerns which had different lid covers and were all slightly different from each other. Two westerns followed one protocol and the other two followed another protocol. When I put the westerns down, I oriented them in a certain way so that I would not accidentally switch the lids. I then had to rinse off the secondary and expose the westerns to ECL in order for the proteins to become visible in the imager. After successfully imaging the westerns, I used the stripping buffer in order to prepare them for the next primary, in order to repeat the process again the next day. I very carefully walked the western blots over to the cold room to allow them to incubate overnight on the rocker.
Next, I had to fix two plates of cells and then do the first day of the cell-staining procedure. The cells were first sitting in media which had a salmon pink color to it. I had to first vacuum out this media and then rinse the cells with PBS and PBSt solution. It was really important not to confuse PBS with PBSt because PBSt contains a detergent which is not in PBS. I also had to treat the plates with Cy3 or FitC depending on the step. I have found that a lot of research is waiting time, which felt a little longer than usual this week with a few less people in the lab than usual due to the 4th of July.
The next day, I repeated the western procedure and reimaged the blots before beginning the second day of the cell- staining procedure. As the plates were now light sensitive, I had to keep them wrapped in tin foil. I have gotten into the habit of leaving them in a drawer by the vacuum when they are not on a rocker in order to keep them out of the way and out of the light. After all of the westerns and the plates were placed on the rocker in the cold room, I extracted DNA from tail samples in order to prepare them for PCR. There were only two samples, but the controls, including no DNA, also had to be put into the PCR machine.
On Wednesday and Thursday, I was able to spend time with my cousin and parents, thanks to 4th of July.
On Friday, I was back in the lab! I added loading dye to my PCR-ed samples before running them in a gel for an hour. During this time, I began the western procedure once again, and continued with day 3 of the cell staining procedure. While the cell plates were incubating, and the westerns were resting on the rocker, I imaged the gel. The image was a little bit streakier than I would have liked, probably due to an excess of DNA being loaded, but I was overall happy with my work.
I was really proud of myself this week for being able to manage a few different things, finding all the necessary chemicals, and for going through the procedures without becoming confused by all of the steps.
Next, I had to fix two plates of cells and then do the first day of the cell-staining procedure. The cells were first sitting in media which had a salmon pink color to it. I had to first vacuum out this media and then rinse the cells with PBS and PBSt solution. It was really important not to confuse PBS with PBSt because PBSt contains a detergent which is not in PBS. I also had to treat the plates with Cy3 or FitC depending on the step. I have found that a lot of research is waiting time, which felt a little longer than usual this week with a few less people in the lab than usual due to the 4th of July.
The next day, I repeated the western procedure and reimaged the blots before beginning the second day of the cell- staining procedure. As the plates were now light sensitive, I had to keep them wrapped in tin foil. I have gotten into the habit of leaving them in a drawer by the vacuum when they are not on a rocker in order to keep them out of the way and out of the light. After all of the westerns and the plates were placed on the rocker in the cold room, I extracted DNA from tail samples in order to prepare them for PCR. There were only two samples, but the controls, including no DNA, also had to be put into the PCR machine.
On Wednesday and Thursday, I was able to spend time with my cousin and parents, thanks to 4th of July.
I was really proud of myself this week for being able to manage a few different things, finding all the necessary chemicals, and for going through the procedures without becoming confused by all of the steps.
Sounds like you did a great job keeping it all organized!
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