Emily Guo, Week 1&2
I've just finished my second week at the lab. The transition has been a bit rough, mostly because the project my graduate student is working on is quite different than what I had prepared for. It also took some time to get situated in New York and to adjust to living on my own.
I'm living in an apartment with three med students in Tower 3, on Haven Avenue. It is about half a mile's walk from my lab and has a nice view, although it is quite noisy at night. As it turns out, my grad student, Alyssa, actually lives in Tower 2, right next door. There are grocery stores and restaurants all along Broadway, two streets over.
Dr. Tycko's lab is located on the sixth floor of the Irving Cancer Research Center on St. Nicholas Avenue:
Everyone usually gets to the lab at 10:00 in the morning, and I usually stay until about 6:00 p.m. The first day, however, I came at 8:00, and had to wait outside for two hours because I didn't have an ID (which I got a few days later). I also had to attend a safety training class the first week. As it turns out, my first day at the lab was also my PI's birthday! He wouldn't open his door when the lab members tried to give him wine.
This is the first time my lab is working with CRISPR/Cas9 technology. We are making cuts in cells from different cell lines to analyze the affects of SNPs with GWAS peaks on methylations and expression. More specifically, we are working with the pSpCas9(BB)-2A-Puro(px459) plasmid to deliver Cas 9 and our guide-RNA into the cells. Alyssa, my grad student, had previously tried introducing Cas9 and gRNA directly into the cells - which is supposed to be the more efficient method - but that had resulted in sub par deletions. When I came along, they were having problems getting the plasmids to take up the insert (none of the E. coli cells were growing in the agar plates, indicating that they did not uptake the ampicillin gene on the plasmid), so my first week involved troubleshooting the procedure for possible solutions.
After a few test runs, we were finally able to successfully grow the E. coli colonies (although there weren't that many)! After consulting various websites and forums, we had decided that Alkaline Phosphatase should only be used for 10 minutes instead of 2 hours to prevent damage to the DNA. Furthermore, using 3-5 ul of our ligation reaction seemed to work much better than the recommended 11 ul.
After extracting the plasmid DNA from the E coli. cells, we ran a diagnostic digest of the plasmids using restriction enzymes that we found on NEB, AflIII and XbaI. Our restriction enzymes would cut the plasmid into a long and short strand (about 8,000 bp & 400 bp respectively). Because we cut out a 20 bp sequence from the plasmid and replaced it with a 25 bp long insert, the plasmid with the insert should have a short strand that is about 5 bp longer than that of the uncut px459 plasmid, a difference that we would be able to see when the two were ran alongside each other on a gel.
Here is a picture of the gel we ran for the the diagnostic digestion of the plasmids. We were a little bit unsure if it would work because the restriction enzymes we found in the freezer were expired, but it turned out fine so we didn't have to order new enzymes. We ran it in 2.5% Metaphor agarose gel for better resolution, and you can see that the smaller strand ran farther in the uncut px459 plasmids. It was very gratifying to see the results after all the work that was done. Now, the pasmid must be sent off for sequencing to make sure the insert did not go through any mutations before it can be used.
Meanwhile, I've been learning different lab techniques, including how to dilute new primers, make different types of gels, gel extraction, designing qPCR primers, finding restriction enzymes, using the nanodrop machine etc. I've never expected that molecular biology labs used so many kits! I'm starting to get a bit more confident and comfortable working in the lab, and I've been given more independent work. My PI had also mentioned showing me how to slice mouse brains sometime...
The lab is relatively small: Anna is another student working at the lab. She's a college student and has had much more lab experience than me, and she's been very helpful in getting me up to speed and familiar with the lab as well as answering my questions. Cathy is the assistant research scientist who does mostly bioinformatics. Jiarwei is a sort of hybrid student/lab technician who is working under Dr. Thomas, one of Dr. Tycko's collaborators who works with stomach cancer, so he is just using the lab space here. Finally, Martha is the lab technician; she's been working with Dr. Tycko for seventeen years.
This week, we found out that Dr. Tycko will be moving his lab to Hackensack in August, where he will have more funding for research. They will probably start moving on August 1st, during my final week at the lab. I'm not sure about how everything is going to work out, and my grad student doesn't yet know if she wants to continue working with Dr. Tycko on the project after the move. This is important because it would affect the direction of the project that we are currently working on.
I'm living in an apartment with three med students in Tower 3, on Haven Avenue. It is about half a mile's walk from my lab and has a nice view, although it is quite noisy at night. As it turns out, my grad student, Alyssa, actually lives in Tower 2, right next door. There are grocery stores and restaurants all along Broadway, two streets over.
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| The Hudson and the George Washington Bridge from my window |
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| Tower 3 (in the back) |
Everyone usually gets to the lab at 10:00 in the morning, and I usually stay until about 6:00 p.m. The first day, however, I came at 8:00, and had to wait outside for two hours because I didn't have an ID (which I got a few days later). I also had to attend a safety training class the first week. As it turns out, my first day at the lab was also my PI's birthday! He wouldn't open his door when the lab members tried to give him wine.
This is the first time my lab is working with CRISPR/Cas9 technology. We are making cuts in cells from different cell lines to analyze the affects of SNPs with GWAS peaks on methylations and expression. More specifically, we are working with the pSpCas9(BB)-2A-Puro(px459) plasmid to deliver Cas 9 and our guide-RNA into the cells. Alyssa, my grad student, had previously tried introducing Cas9 and gRNA directly into the cells - which is supposed to be the more efficient method - but that had resulted in sub par deletions. When I came along, they were having problems getting the plasmids to take up the insert (none of the E. coli cells were growing in the agar plates, indicating that they did not uptake the ampicillin gene on the plasmid), so my first week involved troubleshooting the procedure for possible solutions.
After a few test runs, we were finally able to successfully grow the E. coli colonies (although there weren't that many)! After consulting various websites and forums, we had decided that Alkaline Phosphatase should only be used for 10 minutes instead of 2 hours to prevent damage to the DNA. Furthermore, using 3-5 ul of our ligation reaction seemed to work much better than the recommended 11 ul.
 |
| Our insert/gRNA will go between the BbsI cut sites at the top. |
 |
| Sadly, I didn't take a picture of the successful E. coli plates, but here's the next best thing. |
After extracting the plasmid DNA from the E coli. cells, we ran a diagnostic digest of the plasmids using restriction enzymes that we found on NEB, AflIII and XbaI. Our restriction enzymes would cut the plasmid into a long and short strand (about 8,000 bp & 400 bp respectively). Because we cut out a 20 bp sequence from the plasmid and replaced it with a 25 bp long insert, the plasmid with the insert should have a short strand that is about 5 bp longer than that of the uncut px459 plasmid, a difference that we would be able to see when the two were ran alongside each other on a gel.
Here is a picture of the gel we ran for the the diagnostic digestion of the plasmids. We were a little bit unsure if it would work because the restriction enzymes we found in the freezer were expired, but it turned out fine so we didn't have to order new enzymes. We ran it in 2.5% Metaphor agarose gel for better resolution, and you can see that the smaller strand ran farther in the uncut px459 plasmids. It was very gratifying to see the results after all the work that was done. Now, the pasmid must be sent off for sequencing to make sure the insert did not go through any mutations before it can be used.
Meanwhile, I've been learning different lab techniques, including how to dilute new primers, make different types of gels, gel extraction, designing qPCR primers, finding restriction enzymes, using the nanodrop machine etc. I've never expected that molecular biology labs used so many kits! I'm starting to get a bit more confident and comfortable working in the lab, and I've been given more independent work. My PI had also mentioned showing me how to slice mouse brains sometime...
The lab is relatively small: Anna is another student working at the lab. She's a college student and has had much more lab experience than me, and she's been very helpful in getting me up to speed and familiar with the lab as well as answering my questions. Cathy is the assistant research scientist who does mostly bioinformatics. Jiarwei is a sort of hybrid student/lab technician who is working under Dr. Thomas, one of Dr. Tycko's collaborators who works with stomach cancer, so he is just using the lab space here. Finally, Martha is the lab technician; she's been working with Dr. Tycko for seventeen years.
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| My lab bench |
This week, we found out that Dr. Tycko will be moving his lab to Hackensack in August, where he will have more funding for research. They will probably start moving on August 1st, during my final week at the lab. I'm not sure about how everything is going to work out, and my grad student doesn't yet know if she wants to continue working with Dr. Tycko on the project after the move. This is important because it would affect the direction of the project that we are currently working on.
Sounds like you have been a great help to the lab already! I am sure the upcoming move will be stressful for all involved, hopefully you'll be wrapping up and not too affected.
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