For the past three weeks or so I have been unsuccessfully trying to insert a particular sequence into a plasmid vector containing the HPV16 genome. What makes this particular cloning experiment hard is that the vector is rather large (~10kb!) and my insert is not only small, but it is a mixture of various polymers of the same sequence (anywhere from five to over a hundred repeats). I don't know how many agar plates I went through (maybe twenty?) while I've been trying to get things to work. Jian has been helping me troubleshoot throughout the way to optimize the ligation reaction, DNA extraction methods (for example, is it better to do a gel extraction or the tried and true phenol:chloroform:isoamyl alochol extraction with ethanol precipitation?) It got to the point where I am literally running out of both vector and insert.
Finally, after working on this for weeks, I came in this morning and checked my plates and was surprised to see an explosion of growth! Unfortunately, even my ligation control plate (which is vector without insert) also showed an explosion of growth, so that means that one of the key steps that has been hampering my progress is the de-phosphorylation step with CIP. We ordered new CIP. Fortunately, this is still pretty good since, at first blush, it seems like my ligation reactions still had more growth than the controls. I re-plated my reactions at a much smaller volume, and hopefully they will have reasonably sized colonies for me tomorrow.
It was one of those moments where, after weeks of banging my head against the wall, there was suddenly an "Aha!" moment, and things looked like they are finally falling in place. There is still some work to do to see if there is anything usable, but even if I have to start over, I now have everything in place that it will only take a week to go from start to finish, instead of almost a month. Still, Jian and I have decided that I will stop after this week so I can focus more on reading and preparing my ideas for my thesis project(s). Maybe I can get one of the undergrads to finish things for me. It's kind of nice being able to say that, ha ha.
Monday, March 22, 2010
Monday, March 15, 2010
Thesis Lab Decision
As I am coming to the end of my third and final rotation in Jianxin's lab, I have come to the point where I have to decide which lab I would like to formally join as a full-time grad student.
Initially I thought this decision would be pretty easy; at the conclusion of my rotation with Una last semester, I was 99.999% sure I would join that lab, and I came into Jian's lab with the feeling that I would just enjoy myself and learn as much as I could.
As time progressed in Jian's lab, however, I became really fascinated by the HPV field and some of the unanswered questions, especially mechanisms underlying integration, a critical (and necessary) step for the onset of cervical cancer caused by the virus. And although my rotation project had nothing to do with this, I took time to read more and more and become even more excited about the possibilities. I also found that I was much more actively engaged during lab meetings, offering critical observations about the data and offering my ideas. In short, I have been much more engaged scientifically in Jian's lab than I ever was in Una's lab.
This past week I met with both PI's individually to seriously discuss potential thesis projects, expectations for me as a grad student, things I was interested in or would like to see happen in the lab if I were to join. I finished the week by meeting with Bob Doms, the chair of the microbiology department, and talking to him about my decision and listening to his advice, concerns, and suggestions. It was definitely helpful, and validated my own feelings about my choices and where I thought I wanted to be.
In the end, I decided to join Jian's lab. It feels like the best decision in pretty much every parameter I can think of - funding, research interests, support, etc - but that has not made coming to this decision or moving forward with it easier. My time with Una was fantastic, and it was, by the far, the best environment of people I could ever have hoped for - I hope I will be half as lucky finding labs that felt so much like a family to me and that worked so well together. But in the end, I am here to do research, to investigate and learn, and I think the Jian's lab offers me the best opportunities for all of that.
There are still risks and unknowns in joining Jian's lab: I will be her first graduate student (largely because she is so new here), and that brings with it some uncharted territory for both of us. But this is also an incredible opportunity for me to have a huge impact in the development of the lab, in attracting new students to the lab, in directing where the research goes, etc. There is also the risk in joining a lab with a faculty who hasn't formally received tenure yet. Normally I would stay far, far away from such a lab, because there is the risk the faculty member won't receive tenure while you are in the middle of your graduate work, and generally there is a stronger push to produce papers and get work done in the process of securing tenure. However, in this case, Jian is very well on her way towards receiving tenure and she has had a great team of post-docs who have been helping to establish the lab. Bob (the chair of Microbiology I mentioned above) has no doubt that she will receive tenure, and he probably has the largest voice in that decision out of anyone, so I trust his judgment. I think in this case, the risks involved in joining a junior faculty's lab are much less than other labs I see here at Penn.
I am extremely excited to count myself as Jian's first graduate student, and will be taking the next month or so to really develop my ideas for the questions I am interested in pursuing. Fortunately, a large component of my interest will greatly benefit from Una's expertise in HIV integration, and I have already talked to her about this collaboration. She was extremely excited about it, and I am glad to say that I foresee continuing working with her and her talented crew well into the future.
Initially I thought this decision would be pretty easy; at the conclusion of my rotation with Una last semester, I was 99.999% sure I would join that lab, and I came into Jian's lab with the feeling that I would just enjoy myself and learn as much as I could.
As time progressed in Jian's lab, however, I became really fascinated by the HPV field and some of the unanswered questions, especially mechanisms underlying integration, a critical (and necessary) step for the onset of cervical cancer caused by the virus. And although my rotation project had nothing to do with this, I took time to read more and more and become even more excited about the possibilities. I also found that I was much more actively engaged during lab meetings, offering critical observations about the data and offering my ideas. In short, I have been much more engaged scientifically in Jian's lab than I ever was in Una's lab.
This past week I met with both PI's individually to seriously discuss potential thesis projects, expectations for me as a grad student, things I was interested in or would like to see happen in the lab if I were to join. I finished the week by meeting with Bob Doms, the chair of the microbiology department, and talking to him about my decision and listening to his advice, concerns, and suggestions. It was definitely helpful, and validated my own feelings about my choices and where I thought I wanted to be.
In the end, I decided to join Jian's lab. It feels like the best decision in pretty much every parameter I can think of - funding, research interests, support, etc - but that has not made coming to this decision or moving forward with it easier. My time with Una was fantastic, and it was, by the far, the best environment of people I could ever have hoped for - I hope I will be half as lucky finding labs that felt so much like a family to me and that worked so well together. But in the end, I am here to do research, to investigate and learn, and I think the Jian's lab offers me the best opportunities for all of that.
There are still risks and unknowns in joining Jian's lab: I will be her first graduate student (largely because she is so new here), and that brings with it some uncharted territory for both of us. But this is also an incredible opportunity for me to have a huge impact in the development of the lab, in attracting new students to the lab, in directing where the research goes, etc. There is also the risk in joining a lab with a faculty who hasn't formally received tenure yet. Normally I would stay far, far away from such a lab, because there is the risk the faculty member won't receive tenure while you are in the middle of your graduate work, and generally there is a stronger push to produce papers and get work done in the process of securing tenure. However, in this case, Jian is very well on her way towards receiving tenure and she has had a great team of post-docs who have been helping to establish the lab. Bob (the chair of Microbiology I mentioned above) has no doubt that she will receive tenure, and he probably has the largest voice in that decision out of anyone, so I trust his judgment. I think in this case, the risks involved in joining a junior faculty's lab are much less than other labs I see here at Penn.
I am extremely excited to count myself as Jian's first graduate student, and will be taking the next month or so to really develop my ideas for the questions I am interested in pursuing. Fortunately, a large component of my interest will greatly benefit from Una's expertise in HIV integration, and I have already talked to her about this collaboration. She was extremely excited about it, and I am glad to say that I foresee continuing working with her and her talented crew well into the future.
Tuesday, March 9, 2010
Grad Diary 3/9/10
I'm two days into my "spring break" and so far, it's been nice.
As I mentioned in my previous post, I don't have classes this week (and the undergrads are gone! no long lines at the carts for lunch!). In addition to taking the time to focus on lab stuff, I decided that I would take this week to seriously weigh my options between Una and Jian's lab, and try to come to a decision by the end of the week. To that end I've set up meeting times with both PI's (on different days) to talk seriously about potential thesis projects (both what they are interested as well as my own ideas) as well as wrapping up other concerns/expectations that come with being a full time graduate student.
Bob Doms, the chair for the Microbiology Department, has been very helpful. He works very closely with the graduate students who work in his faculties' labs (mostly MVP students) and meets with them at least once a semester (if not more) to check in, talk about upcoming decisions, etc. A few weeks ago he and I chatted about my vacillation between the two labs, and was happy with the extent and seriousness I have been considering the decision up till now. He did offer some helpful perspective and commentary, and we decided I would check in again when I thought I had made a decision. I will meet with him on Friday to talk about what I feel is the best decision for me (after having met with both PI's) and then make a "formal" announcement to both groups.
To that end I've been making lists and thinking about what the realities might be in both labs, the pros and cons, the differences in lab demeanor and philosophies, the opportunities, and the unknowns (recall that Jian doesn't have any full time grad students...I would be her first). I feel i am coming to a decision already, but I definitely want to take the time to hash it out with both PI's first.
I'll wait until next week to post again with my formal lab decision and how things are moving forward. Wish me luck!
As I mentioned in my previous post, I don't have classes this week (and the undergrads are gone! no long lines at the carts for lunch!). In addition to taking the time to focus on lab stuff, I decided that I would take this week to seriously weigh my options between Una and Jian's lab, and try to come to a decision by the end of the week. To that end I've set up meeting times with both PI's (on different days) to talk seriously about potential thesis projects (both what they are interested as well as my own ideas) as well as wrapping up other concerns/expectations that come with being a full time graduate student.
Bob Doms, the chair for the Microbiology Department, has been very helpful. He works very closely with the graduate students who work in his faculties' labs (mostly MVP students) and meets with them at least once a semester (if not more) to check in, talk about upcoming decisions, etc. A few weeks ago he and I chatted about my vacillation between the two labs, and was happy with the extent and seriousness I have been considering the decision up till now. He did offer some helpful perspective and commentary, and we decided I would check in again when I thought I had made a decision. I will meet with him on Friday to talk about what I feel is the best decision for me (after having met with both PI's) and then make a "formal" announcement to both groups.
To that end I've been making lists and thinking about what the realities might be in both labs, the pros and cons, the differences in lab demeanor and philosophies, the opportunities, and the unknowns (recall that Jian doesn't have any full time grad students...I would be her first). I feel i am coming to a decision already, but I definitely want to take the time to hash it out with both PI's first.
I'll wait until next week to post again with my formal lab decision and how things are moving forward. Wish me luck!
Thursday, March 4, 2010
Grad Diary 3/4/10
Man, these past two days have been tough.
Yesterday morning I had my first Immunology exam, and I think it went well. We were given eight questions and had to answer four. We had an hour and a half, which was just enough time for most people. The questions really varied in style, depth, and approach, leaving something for everyone (I guess). I was a little wary about this test if only because I have never had immunology before, so I tried to be extra careful in preparing and reviewing my notes. I felt pretty good after taking it, and I hope I did well.
This morning was a different story. It was the Gene Regulation exam, and I felt much more prepared for it, having had some background on most of the material already, and the format being similar to the Cell Biology exams I had last semester. The test included six questions, of which we had to answer four, and ideally it should have been doable in one in a half hour. The let us come in an hour early, however, because certain students had a second exam today directly following this one. I came in at the usual time (not feeling a need for a whole extra hour) and was surprised to see pretty much everyone was already there working on it (that shouldn't have surprised me, in retrospect). I picked up my exam, spent a minute or two searching for a free seat and got started. An hour and fifteen minutes later I was done, feeling very fried, and for the first time wishing I had a little more time to feel confident in my answers. I came and left before most anyone else left. Most people used the entire 2.5 hrs. I felt considerably more drained after this exam than after Immunology, and I am not as confident I did as well, but we shall see in a few weeks. Once again my "speedy" test taking was commented on later (though, to be fair, I basically used up the entire time that was supposed to be allotted us anyway).
I'm glad that's all over with and that I can take a week or so to cool off. Next week is "spring break" and I don't have classes next week, but I'll be staying to do lab work. It'll be really nice not having to worry about classes for a few days and just focus on lab stuff and taking it easy. Thank god tomorrow is Friday.
Yesterday morning I had my first Immunology exam, and I think it went well. We were given eight questions and had to answer four. We had an hour and a half, which was just enough time for most people. The questions really varied in style, depth, and approach, leaving something for everyone (I guess). I was a little wary about this test if only because I have never had immunology before, so I tried to be extra careful in preparing and reviewing my notes. I felt pretty good after taking it, and I hope I did well.
This morning was a different story. It was the Gene Regulation exam, and I felt much more prepared for it, having had some background on most of the material already, and the format being similar to the Cell Biology exams I had last semester. The test included six questions, of which we had to answer four, and ideally it should have been doable in one in a half hour. The let us come in an hour early, however, because certain students had a second exam today directly following this one. I came in at the usual time (not feeling a need for a whole extra hour) and was surprised to see pretty much everyone was already there working on it (that shouldn't have surprised me, in retrospect). I picked up my exam, spent a minute or two searching for a free seat and got started. An hour and fifteen minutes later I was done, feeling very fried, and for the first time wishing I had a little more time to feel confident in my answers. I came and left before most anyone else left. Most people used the entire 2.5 hrs. I felt considerably more drained after this exam than after Immunology, and I am not as confident I did as well, but we shall see in a few weeks. Once again my "speedy" test taking was commented on later (though, to be fair, I basically used up the entire time that was supposed to be allotted us anyway).
I'm glad that's all over with and that I can take a week or so to cool off. Next week is "spring break" and I don't have classes next week, but I'll be staying to do lab work. It'll be really nice not having to worry about classes for a few days and just focus on lab stuff and taking it easy. Thank god tomorrow is Friday.
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