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.
Monday, July 30, 2007
Week 6
7/23
Right when I got back from camp, I went straight to work, eager to get going with my experiments. Bryan told me this was going to be a boring week, which I later found out to be true, as I spent most of my time sitting around waiting. Anyway, while I had been away Bryan had moved my Clone A shEGFP cells and my EGFP shEGFP cells to 10cm plates. The third group of cells, Clone A shβ4, had hardly grown (as expected) and had to be moved down into a 24-well plate. Also, I prepared bacteria samples 1.2 and 1.4 for a maxiprep, because we still needed DNA from sample 1. Today I only spent about 45 minutes working in the lab, but had to wait until 2:00 before I could put the bacteria into the incubator so that they wouldn’t overgrow and stop expressing the DNA that we hoped they contained.
7/24
I split all of my 293 samples (4 of them) down 1:20 so that they would be ready for transfection. I then spent the rest of the day performing a maxiprep on samples 1.2 and 1.4, which I hate doing because it involves working with solutions that smell terrible and get everything sticky. Also, it involves over an hour and a half of waiting time, for incubating and centrifuging, which made me glad I brought an ipod. Finally, to make this day officially the worst day yet, after the long process of the maxiprep, neither sample yielded any DNA, and though I was glad to be able to go home, I was upset because I would have to repeat the whole process in two days.
7/25
I plated 2 wells of a 6-well plate with clone A cells to get ready for viral infection, then I replaced the media and added puromycin for selection into the Clone A shβ4 sample. I then replaced the media in 3/4 of the 293 plates with FT media (no antibiotics) and waited until 2:30 this time before I placed the 1.2 and 1.4 cell samples into the incubator for the night.
7/26
I added 2 mL of shβ4 virus to one of the Clone A samples in the 6-well and 2 mL of media to the other sample as a control. I spent the rest of the day maxiprepping the 1.2 and 1.4 samples and once again failed to yield results. Since I was now out of those samples, we decided to not include sample 1 in the rest of the experiment.
7/27
I selected my clone A cells with 10µL/mL puromycin (500µg/mL) and then infected the 293 cells with DNA from samples 2 and 3 using a Lentivirus protocol.
Right when I got back from camp, I went straight to work, eager to get going with my experiments. Bryan told me this was going to be a boring week, which I later found out to be true, as I spent most of my time sitting around waiting. Anyway, while I had been away Bryan had moved my Clone A shEGFP cells and my EGFP shEGFP cells to 10cm plates. The third group of cells, Clone A shβ4, had hardly grown (as expected) and had to be moved down into a 24-well plate. Also, I prepared bacteria samples 1.2 and 1.4 for a maxiprep, because we still needed DNA from sample 1. Today I only spent about 45 minutes working in the lab, but had to wait until 2:00 before I could put the bacteria into the incubator so that they wouldn’t overgrow and stop expressing the DNA that we hoped they contained.
7/24
I split all of my 293 samples (4 of them) down 1:20 so that they would be ready for transfection. I then spent the rest of the day performing a maxiprep on samples 1.2 and 1.4, which I hate doing because it involves working with solutions that smell terrible and get everything sticky. Also, it involves over an hour and a half of waiting time, for incubating and centrifuging, which made me glad I brought an ipod. Finally, to make this day officially the worst day yet, after the long process of the maxiprep, neither sample yielded any DNA, and though I was glad to be able to go home, I was upset because I would have to repeat the whole process in two days.
7/25
I plated 2 wells of a 6-well plate with clone A cells to get ready for viral infection, then I replaced the media and added puromycin for selection into the Clone A shβ4 sample. I then replaced the media in 3/4 of the 293 plates with FT media (no antibiotics) and waited until 2:30 this time before I placed the 1.2 and 1.4 cell samples into the incubator for the night.
7/26
I added 2 mL of shβ4 virus to one of the Clone A samples in the 6-well and 2 mL of media to the other sample as a control. I spent the rest of the day maxiprepping the 1.2 and 1.4 samples and once again failed to yield results. Since I was now out of those samples, we decided to not include sample 1 in the rest of the experiment.
7/27
I selected my clone A cells with 10µL/mL puromycin (500µg/mL) and then infected the 293 cells with DNA from samples 2 and 3 using a Lentivirus protocol.
Monday, July 23, 2007
Week 5
7/9
Today I discovered that, yet again, my bacteria did not grow as I had hoped, so I am starting all over again. I began cloning FSIPPW. Also, I split my clone A cells and EGFP cells onto a six-well dish, with two wells of EGFP at roughly 6x10^5 and three wells of clone A cells at roughly 6x10^5. I also split my 293 cells.
7/10
I went through the CIP treatment, annealing, gel extraction, phosphorylation, ligation, and transformation steps again. However, I began harvesting my virus by inserting the virus into plates of cells, some with Clone A cells and others with EGFP expressing cells.
7/11
Today was another repeat day. I noticed that 2/3 of my bacteria looked great. Sample #2, however, looked questionable. First, however, I added .5mg/ml puromycin to my virus infected cells, and then froze down the extra virus. To tell you the truth, I am getting pretty sick of viruses and how many precautions I must take. The only difference between this virus and my first one is that this one can kill me. Taking this into consideration, I decided to follow proper procedure and soak anything that the virus could have possibly infected in my beaker of bleach. I also got some great pictures of me in my lab coat (though it was way too big on me.)
At the end of the day, I inserted one colony of bacteria into each of 15 tubes, five from each sample. I can only hope that this time they will express the DNA.
7/12
I repeated the entire miniprep day on my bacteria to purify the DNA. I then ran the DNA through an agarose gel, examined it under a UVP light . . . SUCCESS!!! this time I had six out of the 12 colonies (I accidentally contaminated three of my samples) express the segments I had inserted. I picked the three strongest expressing samples and grew them overnight in a 37º high-speed shaker.
7/13
I began the day by splitting my clone A cells, EGFP expressing cells, and 293 cells 1:20—I will be away for a week and don’t want them to overgrow. I then moved my virus expressing cells onto 10 cm plates. I spent the entire day repeating a maxiprep on my bacteria. Every step was performed carefully, double-checking every instruction. Brian told me that if it did not work this time, it would be too late to start over and I would have to find something else to work on. Anticipation was controlling me as I performed my final centrifuge spin of the day. As I removed the supernatant and watched the interior of the tube slowly dry in the open air, I noticed on the bottom of the tubes a clear glob of what I could only hope was purified DNA. I was worried because I did not see this glob on the tube containing what I thought would be DNA sample 1. I was practically sweating as I ran the samples through the photometer. I first ran a blank control first, which printed out as 0.000 µg/ml. Then sample 1, as the machine buzzed and beeped I prayed for good news, only to see a 0.000 µg/ml dsDNA come out of the printer. Now I was scared. As I loaded sample 2 I could sense my project failing . . . but then, success. The printer astonished both me and Brian at the number. 6605 µg/ml dsDNA. He informed me that this was a lot more DNA than he expected and when the third sample printed out a 4670.4 µg/ml I knew that it was no fluke. I happily froze my DNA in the –20º freezer and went home. It turns out that Friday the 13th was my lucky day after all.
Today I discovered that, yet again, my bacteria did not grow as I had hoped, so I am starting all over again. I began cloning FSIPPW. Also, I split my clone A cells and EGFP cells onto a six-well dish, with two wells of EGFP at roughly 6x10^5 and three wells of clone A cells at roughly 6x10^5. I also split my 293 cells.
7/10
I went through the CIP treatment, annealing, gel extraction, phosphorylation, ligation, and transformation steps again. However, I began harvesting my virus by inserting the virus into plates of cells, some with Clone A cells and others with EGFP expressing cells.
7/11
Today was another repeat day. I noticed that 2/3 of my bacteria looked great. Sample #2, however, looked questionable. First, however, I added .5mg/ml puromycin to my virus infected cells, and then froze down the extra virus. To tell you the truth, I am getting pretty sick of viruses and how many precautions I must take. The only difference between this virus and my first one is that this one can kill me. Taking this into consideration, I decided to follow proper procedure and soak anything that the virus could have possibly infected in my beaker of bleach. I also got some great pictures of me in my lab coat (though it was way too big on me.)
At the end of the day, I inserted one colony of bacteria into each of 15 tubes, five from each sample. I can only hope that this time they will express the DNA.
7/12
I repeated the entire miniprep day on my bacteria to purify the DNA. I then ran the DNA through an agarose gel, examined it under a UVP light . . . SUCCESS!!! this time I had six out of the 12 colonies (I accidentally contaminated three of my samples) express the segments I had inserted. I picked the three strongest expressing samples and grew them overnight in a 37º high-speed shaker.
7/13
I began the day by splitting my clone A cells, EGFP expressing cells, and 293 cells 1:20—I will be away for a week and don’t want them to overgrow. I then moved my virus expressing cells onto 10 cm plates. I spent the entire day repeating a maxiprep on my bacteria. Every step was performed carefully, double-checking every instruction. Brian told me that if it did not work this time, it would be too late to start over and I would have to find something else to work on. Anticipation was controlling me as I performed my final centrifuge spin of the day. As I removed the supernatant and watched the interior of the tube slowly dry in the open air, I noticed on the bottom of the tubes a clear glob of what I could only hope was purified DNA. I was worried because I did not see this glob on the tube containing what I thought would be DNA sample 1. I was practically sweating as I ran the samples through the photometer. I first ran a blank control first, which printed out as 0.000 µg/ml. Then sample 1, as the machine buzzed and beeped I prayed for good news, only to see a 0.000 µg/ml dsDNA come out of the printer. Now I was scared. As I loaded sample 2 I could sense my project failing . . . but then, success. The printer astonished both me and Brian at the number. 6605 µg/ml dsDNA. He informed me that this was a lot more DNA than he expected and when the third sample printed out a 4670.4 µg/ml I knew that it was no fluke. I happily froze my DNA in the –20º freezer and went home. It turns out that Friday the 13th was my lucky day after all.
Monday, July 9, 2007
Week 4
7/5
I began cloning FSIPPW again.
I added 5 x 106 293 cells onto plates, 5 million on each of four plates with FT media (no antibiotics). I then added 500,000 cells onto an additional plate to grow.
7/6
Today was a really busy day, but you have heard it all before. I annealed the oligos again, CIP treated the FSIPPW, split my clone A cells, ligated the FSIPPW and oligos, then transformed my bacteria.
I began cloning FSIPPW again.
I added 5 x 106 293 cells onto plates, 5 million on each of four plates with FT media (no antibiotics). I then added 500,000 cells onto an additional plate to grow.
7/6
Today was a really busy day, but you have heard it all before. I annealed the oligos again, CIP treated the FSIPPW, split my clone A cells, ligated the FSIPPW and oligos, then transformed my bacteria.
Week 3
6/25
Today I counted and split my 293 FT cells, putting roughly 3 million cells into each of three dishes. I then replaced the media in my virus/control cells with 1µg/µL of G418. I split my clone A cells 1:10 and that was it for the day.
6/26
I began making a virus today. I will list the reagents as follows:
Tube Ingredients
A- β4 FSIPPW .41µg/µL= 7.317µL + 9µL Packaging + 1.5mL Opti-Mem media
B- EGFP FSIPPW .904µg/µL= 3.319µL + 9µL Packaging +1.5mL Opti-Mem media
C- 3mL Opti-Mem 72µL Lipofectamine
I waited 5 minutes for the Lipofectamine to incubate, then added 1.5mL of tube C to each other tube, then incubated at room temperature for 20 minutes. Then I replaced the FT media in the plates with 5mL of Opti-Mem media and incubated at 37º for 20 minutes. I then aspirated the media out of the plates and added 3mL of tube A into 1 plate and 3mL of tube B into the other plate and incubated at 37º for 5 hours. Then I added 5mL of FT media (no serum or antibiotics) with 20% FBS to the plates and incubated them at 37º overnight.
Next, I began what is called a Miniprep kit on my bacteria. I spun down 4mL of each tube (15 of them) at 4000 rpm for 5 minutes. I then followed the instructions of the miniprep kit down to the letter, which essentially just isolated the DNA that had been growing inside of the E. Coli. I then digested the Minipreps using the enzymes EcoRI and BamHI and then incubated at 37º for 90 minutes. I ran the digest mix through a 2% agarose gel and picked the three best looking samples after analyzing under a UVP. Sadly, none of the samples yielded the results I was looking for, so I was forced to hope that the DNA I was trying to find was just in too low concentrations to be picked up. I made three 250mL flasks of LB/amp with the samples added (500µL of each) and put them in a high-speed shaker at 37º overnight.
6/27
I made a batch of serum and antibiotic-free FT media, adding 1mM Sodium Pyruvate, 1mM NEAA, and 2mM L-Glutamine. I then replaced the media in the 293 cells with 10mL of 10% FBS DMEM (serum and antibiotic free)
I ran the DNA I had been growing through a Maxiprep kit, which gave me higher concentrations of the DNA. I digested them overnight along with a control FSIPPW using the miniprep reagents.
6/28
I removed the digested lysates from the incubator and ran them on a 2% agarose gel again at 110 volts. I again viewed the gel under a UVP and the results came back negative. This means that I need to redo the experiment all over again.
I split my clone A cells 2:5 and froze down my eGFP cells. I took Friday off and left for wrestling camp on Saturday, not to return until July 5th.
Today I counted and split my 293 FT cells, putting roughly 3 million cells into each of three dishes. I then replaced the media in my virus/control cells with 1µg/µL of G418. I split my clone A cells 1:10 and that was it for the day.
6/26
I began making a virus today. I will list the reagents as follows:
Tube Ingredients
A- β4 FSIPPW .41µg/µL= 7.317µL + 9µL Packaging + 1.5mL Opti-Mem media
B- EGFP FSIPPW .904µg/µL= 3.319µL + 9µL Packaging +1.5mL Opti-Mem media
C- 3mL Opti-Mem 72µL Lipofectamine
I waited 5 minutes for the Lipofectamine to incubate, then added 1.5mL of tube C to each other tube, then incubated at room temperature for 20 minutes. Then I replaced the FT media in the plates with 5mL of Opti-Mem media and incubated at 37º for 20 minutes. I then aspirated the media out of the plates and added 3mL of tube A into 1 plate and 3mL of tube B into the other plate and incubated at 37º for 5 hours. Then I added 5mL of FT media (no serum or antibiotics) with 20% FBS to the plates and incubated them at 37º overnight.
Next, I began what is called a Miniprep kit on my bacteria. I spun down 4mL of each tube (15 of them) at 4000 rpm for 5 minutes. I then followed the instructions of the miniprep kit down to the letter, which essentially just isolated the DNA that had been growing inside of the E. Coli. I then digested the Minipreps using the enzymes EcoRI and BamHI and then incubated at 37º for 90 minutes. I ran the digest mix through a 2% agarose gel and picked the three best looking samples after analyzing under a UVP. Sadly, none of the samples yielded the results I was looking for, so I was forced to hope that the DNA I was trying to find was just in too low concentrations to be picked up. I made three 250mL flasks of LB/amp with the samples added (500µL of each) and put them in a high-speed shaker at 37º overnight.
6/27
I made a batch of serum and antibiotic-free FT media, adding 1mM Sodium Pyruvate, 1mM NEAA, and 2mM L-Glutamine. I then replaced the media in the 293 cells with 10mL of 10% FBS DMEM (serum and antibiotic free)
I ran the DNA I had been growing through a Maxiprep kit, which gave me higher concentrations of the DNA. I digested them overnight along with a control FSIPPW using the miniprep reagents.
6/28
I removed the digested lysates from the incubator and ran them on a 2% agarose gel again at 110 volts. I again viewed the gel under a UVP and the results came back negative. This means that I need to redo the experiment all over again.
I split my clone A cells 2:5 and froze down my eGFP cells. I took Friday off and left for wrestling camp on Saturday, not to return until July 5th.
Friday, July 6, 2007
Week 2
6/18
I began the week by taking another look at my death curve in case anything changed over the weekend. Luckily, the results remained unchanged and I was now certain of the optimal ratio of puromycin to use. Then, I began my first cell count. A cell count consists of using simple ratios to count the approximate number of cells in a culture plate. First, I resuspended the cells on my plate into 8mL of growth media. Then I removed 10µL of the media and mixed it with 10µL of special stain. I took 10µL of this stain and spread it onto a glass plate, which contained a grid pattern. Using a microscope, I counted the number of cells on four of the grids on the plate and ended up with the number 191. I multiplied 191 by 2 (a 1:1 dilution) and then divided the result (382) by 4 (four grids). Because of the way the glass plate is designed, this meant there were 95.5 x 104 cells (950,000) in 1mL of media. Because there was roughly 7.5mL of media, I multiplied my number by 7.5 and concluded that there were 7,100,000 cells in the media. I then resuspended my cells in 7.1mL of media, so that each mL of media should contain 1 million cells.
The special RNA I ordered came in today, and that allowed me to begin work on the second phase of my project. I started to clone an FSIPPW DNA backbone, first by digesting it. I added 8.34µL of FSIPPW, 25.66µL H20, 4µL EcoRI Buffer, 1µL EcoRI, and 1µL BamHI. I placed the backbone and enzymes in a 37 degree water bath overnight.
I also resuspended 293-T cells in FT media and left it to incubate overnight at 37 degrees
Then I suspended the special oligos I ordered in RNAse-free water so the concentration was .1nM
6/19
This morning I added 1µL of CIP to digest the FSIPPW mixture. I incubated that at 37 degrees for 1 hour. Then I added 8µL of EDTA to inactivate the CIP, and left that at 75 degrees for 10 minutes. I ran the mixture through a 1% agarose gel with a 1kbp DNA marker. I later extracted a glowing band (visible with UV light) and eluted the pure DNA with a Qiagen gel extraction kit (this took forever).
I annealed the oligos I suspended the day before with a PCR program called Liz oligo, which consists of 95 degrees for 10 minutes, 70 degrees for 10 minutes, decreasing 1 degree/minute until it reaches 25 degrees. Phosphorylating these oligos consisted of adding the oligos to T4PNK and ATP, which I incubated at 37 degrees for 30 minutes, then 70 degrees for 10 minutes, then put on ice. I then began the Ligation process, adding the oligos to the purified FSIPPW and ligase, with a negative control that used water instead of the Oligos.
Transformation consisted of preparing LB media by adding MgC12, MgSO4, and glucose. I mixed 3.4µL B-ME per 200 µL aliquot XL-1 blue competent cells into a 1.5 mL tube. Then, after adding 1 µL of the ligation mix to each tube, I incubated them on ice for 30 minutes, heat shocked them at 42 degrees for 90 seconds, and then put them back on ice for 2 minutes before adding the cell mixture to 1 mL of warmed media and shaking at 37 degrees for 45 minutes. I then resuspended in 100µL of media and spread on a LB/Amp plate.
I froze one of my plates of 293-T cells in a cryogenic freezer (liquid nitrogen tank) and then split my other stock onto a plate and added G418 “neomycin.”
With my clone A cells, I added polybrene to the 6-well plate, then added the retrovirus. One thing I did learn about working with viruses, however, is that people are paranoid of what they can do. I worked with a retrovirus today, like HIV/AIDS, but the only effects are a slight immunity to neomycin and glowing green under a certain light. I had to have a beaker of bleach next to me at all times, and everything that touched what could have virus on it had to be rinsed and then ejected into bleach. The end result was a beaker filled more with pipet tips than with the bleach itself. After this, I rinsed everything with bleach and then put the cells in the incubator.
6/20
Oh, man…
The colonies did not grow on the LB/Amp plates, so I repeated the transformation step of yesterday’s experiment, adding twice the amount of cells to the plates as last time. Also, instead of shaking the bacteria for 45 minutes, I left them in there for three hours so they had more time to recover. I then began digesting another batch of FSIPPW in case the E. Coli still did not grow.
I removed the virus from the cell plate and replaced the media with regular growth media, then placed the cells back in the incubator.
Since today was a slow day, I helped Brian with some clone A cells of his. I added different secondary stains to his cells, some receiving Fascin 1:50, others Fascin 1:100, a third group Mouse IgG, and a fourth Phalloidin. Then I proceeded to perform a long and rather annoying wash-rinse-repeat cycle using TBS-T and PBS.
6/21
Bad news for me . . .
The colonies still did not grow, so I repeated all of the experiments that I did on the 19th. Then I mounted cover slips with Brian on the cells I had rinsed yesterday.
6/22
Finally!
The E. Coli had grown, so I picked five of the best colonies from each tray and isolated them in their own media. I then put them in a 37 degree high-speed shaker overnight. Then I went back to helping Brian.
I performed a task known as rtPCR with a Sox 9 protein. To make a really long story short, we performed a PCR on the protein and later, examining the gel, we found a band around where we wanted one to be, so I wasn’t really sure what that meant but he seemed pretty clear that it was good news.
I counted and split my 293 cells, then replaced the media in the virus/control cells with 1µg/mL G418. Finally, I split my clone A cells and went home for the weekend.
I began the week by taking another look at my death curve in case anything changed over the weekend. Luckily, the results remained unchanged and I was now certain of the optimal ratio of puromycin to use. Then, I began my first cell count. A cell count consists of using simple ratios to count the approximate number of cells in a culture plate. First, I resuspended the cells on my plate into 8mL of growth media. Then I removed 10µL of the media and mixed it with 10µL of special stain. I took 10µL of this stain and spread it onto a glass plate, which contained a grid pattern. Using a microscope, I counted the number of cells on four of the grids on the plate and ended up with the number 191. I multiplied 191 by 2 (a 1:1 dilution) and then divided the result (382) by 4 (four grids). Because of the way the glass plate is designed, this meant there were 95.5 x 104 cells (950,000) in 1mL of media. Because there was roughly 7.5mL of media, I multiplied my number by 7.5 and concluded that there were 7,100,000 cells in the media. I then resuspended my cells in 7.1mL of media, so that each mL of media should contain 1 million cells.
The special RNA I ordered came in today, and that allowed me to begin work on the second phase of my project. I started to clone an FSIPPW DNA backbone, first by digesting it. I added 8.34µL of FSIPPW, 25.66µL H20, 4µL EcoRI Buffer, 1µL EcoRI, and 1µL BamHI. I placed the backbone and enzymes in a 37 degree water bath overnight.
I also resuspended 293-T cells in FT media and left it to incubate overnight at 37 degrees
Then I suspended the special oligos I ordered in RNAse-free water so the concentration was .1nM
6/19
This morning I added 1µL of CIP to digest the FSIPPW mixture. I incubated that at 37 degrees for 1 hour. Then I added 8µL of EDTA to inactivate the CIP, and left that at 75 degrees for 10 minutes. I ran the mixture through a 1% agarose gel with a 1kbp DNA marker. I later extracted a glowing band (visible with UV light) and eluted the pure DNA with a Qiagen gel extraction kit (this took forever).
I annealed the oligos I suspended the day before with a PCR program called Liz oligo, which consists of 95 degrees for 10 minutes, 70 degrees for 10 minutes, decreasing 1 degree/minute until it reaches 25 degrees. Phosphorylating these oligos consisted of adding the oligos to T4PNK and ATP, which I incubated at 37 degrees for 30 minutes, then 70 degrees for 10 minutes, then put on ice. I then began the Ligation process, adding the oligos to the purified FSIPPW and ligase, with a negative control that used water instead of the Oligos.
Transformation consisted of preparing LB media by adding MgC12, MgSO4, and glucose. I mixed 3.4µL B-ME per 200 µL aliquot XL-1 blue competent cells into a 1.5 mL tube. Then, after adding 1 µL of the ligation mix to each tube, I incubated them on ice for 30 minutes, heat shocked them at 42 degrees for 90 seconds, and then put them back on ice for 2 minutes before adding the cell mixture to 1 mL of warmed media and shaking at 37 degrees for 45 minutes. I then resuspended in 100µL of media and spread on a LB/Amp plate.
I froze one of my plates of 293-T cells in a cryogenic freezer (liquid nitrogen tank) and then split my other stock onto a plate and added G418 “neomycin.”
With my clone A cells, I added polybrene to the 6-well plate, then added the retrovirus. One thing I did learn about working with viruses, however, is that people are paranoid of what they can do. I worked with a retrovirus today, like HIV/AIDS, but the only effects are a slight immunity to neomycin and glowing green under a certain light. I had to have a beaker of bleach next to me at all times, and everything that touched what could have virus on it had to be rinsed and then ejected into bleach. The end result was a beaker filled more with pipet tips than with the bleach itself. After this, I rinsed everything with bleach and then put the cells in the incubator.
6/20
Oh, man…
The colonies did not grow on the LB/Amp plates, so I repeated the transformation step of yesterday’s experiment, adding twice the amount of cells to the plates as last time. Also, instead of shaking the bacteria for 45 minutes, I left them in there for three hours so they had more time to recover. I then began digesting another batch of FSIPPW in case the E. Coli still did not grow.
I removed the virus from the cell plate and replaced the media with regular growth media, then placed the cells back in the incubator.
Since today was a slow day, I helped Brian with some clone A cells of his. I added different secondary stains to his cells, some receiving Fascin 1:50, others Fascin 1:100, a third group Mouse IgG, and a fourth Phalloidin. Then I proceeded to perform a long and rather annoying wash-rinse-repeat cycle using TBS-T and PBS.
6/21
Bad news for me . . .
The colonies still did not grow, so I repeated all of the experiments that I did on the 19th. Then I mounted cover slips with Brian on the cells I had rinsed yesterday.
6/22
Finally!
The E. Coli had grown, so I picked five of the best colonies from each tray and isolated them in their own media. I then put them in a 37 degree high-speed shaker overnight. Then I went back to helping Brian.
I performed a task known as rtPCR with a Sox 9 protein. To make a really long story short, we performed a PCR on the protein and later, examining the gel, we found a band around where we wanted one to be, so I wasn’t really sure what that meant but he seemed pretty clear that it was good news.
I counted and split my 293 cells, then replaced the media in the virus/control cells with 1µg/mL G418. Finally, I split my clone A cells and went home for the weekend.
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