8/6
At the start of my final week, I begin by taking pictures of the cover slides that I made. We took pictures of cells with F-Actin staining—this was to make sure that the shEGFP did not affect the cytoskeleton in any way and allowed them to be used as a control. I split down more of my cells and replaced the media in all of them. I also plated 3mL of EGFP expressing cells with shEGFP and Clone A cells with shEGFP in a 6-well plate. I then extracted and purified RNA and protein lysates from 3.2 virus cells, Clone A cells, and Clone A shEGFP cells the same way I did the previous week.
8/7
Bad news today. I put the RNA through PCR and later, when they came out, I checked them under the UVP machine for any β4 knockdown. Unfortunately, the β4 did not appear knocked down at the RNA level, so my only hope was for the cell lysates to show a β4 knockdown. I began to develop the lysates, running them through an 8% gel and then coating them with 505 antibody overnight. Before I left, I coated 12 wells of a 24 well plate with laminin for a migration assay.
8/8
Right when I came in today I began developing my Western Blot. I washed off the antibody and then applied a secondary anti-rabbit antibody. Once this was washed off, I applied the luminescent developing material and then took the membrane to the dark room. Waiting the 5 minutes for the film to come out of the machine seemed like the longest 5 minutes of my life. The film finally rolled out of the developing machine and I could see that all my hard work had finally paid off. The β4 levels in the protein of the cells containing my virus have dropped dramatically.
Since my experiment finally received some good data, we began to hurry on the migration assay because my presentation is tomorrow and I need some conclusions. I spent hours waiting for my migration plate to finish incubating after I had coated the wells with special media and added my cells, then I anxiously washed the cells and applied the luminescent developing chemicals. We hurried to the microscope and saw that the β4 migration was only slightly reduced, giving me inconclusive results. Still, my experiment proved a success, even without concrete results. I had successfully knocked down the β4 levels in Clone A cells, which is all that mattered.
8/9
Today is my last day. I gave my PowerPoint presentation to the lab, which I will also present at Brooks in the fall. After going over my data and results, I cleaned up my area and we said our goodbyes. Thus concludes my experience at UMASS Medical Center.
Tuesday, August 21, 2007
Week 7
7/30
Today I finished producing my virus, meaning that the DNA from samples two and three was now ready to infect the Clone A cells. The rest of the day I performed basic cell maintenance on all of my cells, which consists of splitting them down and/or replacing the media. I spread some of my Clone A cells onto smaller 60mm plates to get them ready for infection.
7/31
Today was a long day. I started by purifying the RNA and proteins from my cells, specifically the Clone As, both normal and with shEGFP, and also the EGFP expressing cells, both normal and with shEGFP. I followed a kit called Qiagen Rneasy and was able to extract and purify the RNA from the cells and observed them with a biophotometer, which gave me great results. The proteins were simpler. I just added a special lysis buffer that I made and kept them on ice for 1 hour, vortexing them every 10 minutes. Once that was done, I spun the solution in a microcentrifuge for 10 minutes and then collected the supernatant. I analyzed the results through a biophotometer and saw that I had relatively large amounts of protein from all but the Clone A shEGFP cells. At the end of the long day, I replaced the media of my Clone A cells with special virus containing media, letting the cells be transfected over the next two days.
8/1
I started selection for my Clone A cells today, adding 5mg of puromycin to the plates with a replacement of media so that cells not expressing the virus would be killed. I took my protein samples and ran them through an 8% gel so that I could observe the effects of EGFP on β4 through a western blot. This task takes two full days, as the transfer membranes needed to sit overnight in milk containing antibodies. Also today, I put my RNA through PCR so that it would be DNA by the next morning.
8/2
I got in early this morning and began washing the Western blot membranes. Then I had them sit in milk with a secondary anti-rabbit antibody for 45 minutes. After that, I developed the film and saw that I had somehow reduced β4 expression in my shEGFP cells. We ended up interpreting these results differently because when we examined the cells, we found that I had accidentally poisoned them a few days ago by adding G418 instead of puromycin. Moving on, we ran the PCR results through a 1% gel in order to analyze the results. The results weren’t conclusive, but since that whole process was mostly done for practice, we moved on with coating cover slips with approximately 100,000 cells each, to get them ready for photographing the next day.
8/3
I spent most of the day adding various antibodies to the cover slips, to get them to express different proteins under luminescent photography. I also went through more cell maintenance before mounting the cover slips onto slides at the end of the day.
Today I finished producing my virus, meaning that the DNA from samples two and three was now ready to infect the Clone A cells. The rest of the day I performed basic cell maintenance on all of my cells, which consists of splitting them down and/or replacing the media. I spread some of my Clone A cells onto smaller 60mm plates to get them ready for infection.
7/31
Today was a long day. I started by purifying the RNA and proteins from my cells, specifically the Clone As, both normal and with shEGFP, and also the EGFP expressing cells, both normal and with shEGFP. I followed a kit called Qiagen Rneasy and was able to extract and purify the RNA from the cells and observed them with a biophotometer, which gave me great results. The proteins were simpler. I just added a special lysis buffer that I made and kept them on ice for 1 hour, vortexing them every 10 minutes. Once that was done, I spun the solution in a microcentrifuge for 10 minutes and then collected the supernatant. I analyzed the results through a biophotometer and saw that I had relatively large amounts of protein from all but the Clone A shEGFP cells. At the end of the long day, I replaced the media of my Clone A cells with special virus containing media, letting the cells be transfected over the next two days.
8/1
I started selection for my Clone A cells today, adding 5mg of puromycin to the plates with a replacement of media so that cells not expressing the virus would be killed. I took my protein samples and ran them through an 8% gel so that I could observe the effects of EGFP on β4 through a western blot. This task takes two full days, as the transfer membranes needed to sit overnight in milk containing antibodies. Also today, I put my RNA through PCR so that it would be DNA by the next morning.
8/2
I got in early this morning and began washing the Western blot membranes. Then I had them sit in milk with a secondary anti-rabbit antibody for 45 minutes. After that, I developed the film and saw that I had somehow reduced β4 expression in my shEGFP cells. We ended up interpreting these results differently because when we examined the cells, we found that I had accidentally poisoned them a few days ago by adding G418 instead of puromycin. Moving on, we ran the PCR results through a 1% gel in order to analyze the results. The results weren’t conclusive, but since that whole process was mostly done for practice, we moved on with coating cover slips with approximately 100,000 cells each, to get them ready for photographing the next day.
8/3
I spent most of the day adding various antibodies to the cover slips, to get them to express different proteins under luminescent photography. I also went through more cell maintenance before mounting the cover slips onto slides at the end of the day.
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