1.28.15, Wednesday, Today I wanted to pull my hair out
Deciphering plasmid maps. This morning, I tried to decipher the sequencing data and plasmid maps the Martinez lab had sent me. I hope they don't read my blog. They're in Austria and publish in EMBOJ - a journal with an impact rating of 10 something. They sound so smart. Anyway. Most of it made sense, but there were some discrepancies that were bothering me. I went to ask Becky about them - and she said - yes - those were serious questions. I needed to confirm with them exactly what the vectors are, so that I can potentially do the cloning I want to do.
Cloning plans ... to take over the world. I'm going to learn to clone. I've had this vandetta for a while. Melissa taught me how to do it. I recited the steps to Becky and she said I was correct. But that was when I was an undergrad. I just need to learn the "grad school details." Because when you're a grad student and have to do everything by yourself, you discover that you didn't know half the details you needed to know to do it - those things your mentor told you that you didn't have to figure out for yourself as an undergrad.
I want to clone the RtcB gene I have from a mammalian to an E.coli vector, so that I can express it in E.coli. And I want to do it myself. ... I'm ... not really sure why. I just have this urge. Kind of like ... *steeples fingers* *strokes non-existent beard* ... "I bet I could do that in a week." *nods to self with mischievous, greedy glint in eyes.* Don't ask me. Am I challenging myself? Maybe. My logical side realizes that is stupid and I'm imagining this fantasy world where everything works right the first time, but my stubborn side doesn't care. It's probably made worse by the fact that I feel like I'm not moving and am desperate to be. Becky says that's it's a really nice skill to have on your resume, and SHE is willing to teach me. SO THERE. If it doesn't work in 2 weeks, I'll give up and give it to Oskar (the guy who we pay to clone stuff). ... Am I trying to give myself a hard time and make things harder for myself?? Ahhhhhhhhhrrrrrrggg, I've no idea. It just irritates me that I'm not doing it myself. I like to be independent and do everything myself. Except for sequencing. I don't mind if Genewiz does that. That's what it is. I like to be independent. ... [argues with self uselessly for way too many more words]
Random thoughts about my protein. I worried that human RtcB couldn't express in E.coli due to size, but that isn't so. It's not that big - no bigger than E.coli version or P.horikoshii. It should work. We shall see!
Sending in plasmids for sequencing. After talking to Becky, I finally got my plasmids sent out for sequencing, after guessing as to their size. It wasn't written anywhere conclusively in the data I was sent, and I couldn't find the exact gene insert, except for one. So I guessed. Becky said that was ok. Genewiz would still sequence them.
MALDI. The Beast. Then Sam came in. During the day, we tried to run more MALDI, found out some mistakes that needed to be corrected, and more information about how MALDI works. I need to read more about MALDI. I feel like a MALDI idiot. MALDI has yet to work one time for me in my life so far. I need to understand how it works better. For some reason ... it doesn't go in ... or whatever, I don't know.
Experiment Sam and I thought about doing but didn't. We were going to run a transformation, but didn't end up having time for it, because I was going to use some of Kornelia's cells, and she wasn't there at the time to show me where they were. I called her, but she wanted to show me in person so she could check that the stocks were viable. That's ok. That experiment can wait. I just want to transform the human archease pET plasmid into an E.coli expression strain so I can express that soon. I'll probably try one day expression again. Then, I'll feel better. I like to be moving, doing, going, experimenting! Sitting at my desk, reading, stressing, deciphering, organizing tends to raise my stress levels really fast if I do that for several days in a row like I have been.
Sam's protein gel. Then, I also showed Sam how to image his protein gel. It's too faint. Grr. But we found out that if you don't include loading dye in the gel samples, it still runs ok. That isn't true for DNA / RNA gels - glycerol in the dye is necessary for the sample to sink into the well. So that was interesting to find out. (I forgot to tell him to include that yesterday in my rush to get to class.) Kornelia was interested in that result as well. We almost also dried the gel too, but didn't get to it. By the time we could have, it was 6 pm, and I was oozing stress, and I just couldn't stay a second longer. I told John I wanted to eat a huge cup of chocolate fudge coffee with pizza and watch a violent movie. That's kind of how I felt. But now I'm writing this long post. And I'm feeling better. If you want to know why I'm writing this long post - that's why. Writing relaxes me. Most of the time.
Getting ready for Sam's PH RtcB activity assays! Sam and I were discussing his next steps. The activity assays! And I was like - oh yay! - how exciting! I'll get Sam working on this and it'll be great - if I can't get my experiments going, I'll get HIS going! ... oh yeah ... the BUFFER PH QUESTION!! *headdesk* *headdesk* *headdesk* I suddenly remembered the problem I ran into when I wanted to do the activity assays a few weeks ago. The buffer needs to be pHed at 70C. Buffers tend to vary their pH with temperature. And it's an RNA buffer. That makes it 1000x worse to pH. *sigh* I explained what would have to be done to Sam, and he groaned. Suddenly, I'm glad I'm not doing that experiment. Just planning it really shot up my stress levels. I tried to write a protocol for him, find all the things he'd need to do / know, calculate what it would take to make a new GTP stock, and I about had a heart attack. I never finished that. I'll do that first thing in the morning.
Other conversations: Spencer. In the morning, while looking for Becky, I instead found Spencer, and asked him about his project. Got to hear an update on that and that was cool. Told him about mine. Hope I didn't keep him too long. I'm starting to become a fixture in the Conticello lab now too. That makes the fourth lab I'm starting to haunt simultaneously. I'm becoming more quantum mechanical by the moment.(Dr. Lynn called me a quantum mechanical vagabond, because I was everywhere at once, he said.)
Other conversations: Yimu. I met Yimu in the hall in the morning at 8 am. She is in the Hill lab next to me and was getting coffee so I gave her some of mine. She said she knew just how I felt, about being stuck in the mud. She was having to redo all her experiments using a different material of electrode, and she was trying to figure out which material to choose. She's so sweet and nice to talk to. Just talking to her lowers my stress. She also laughs a lot.
Kevin being Kevin. Kevin's presence in the lab was soothing. He broke into dance and song twice today. Once he sang about "serenading me with his voice," in his horrible, hilariously funny, awesome singing style. Then later he sang about the MALDI plate being missing and Sam and I finding it, saying it was under a pot of gold at the end of a rainbow. And he moonwalked for us. And told us how awesome it would be if he fell over a chair and hurt himself while doing that. In the evening, he told us he was helping Yuan's student grow up faster. At which point I told him I'd skewer him with my lightsaber. And he did this all while Nanodropping and doing experiments with his particles. I'm going to seriously miss Kevin. He's the kind of mischievous person you might not always entirely agree with but can't help loving to death anyway.
Home. I got home at 7:15 pm and wrote rapidly. Then ate voraciously. Then wrote rapidly some more. I feel much better now. At 8:21 pm, I'm about to go to bed. Here's a typical schedule of what I usually do. I doubt it's the typical grad school schedule, but I do know of some early birds.
5:30 am - alarm goes off - wake up - curl up next to John
5:35 am - alarm goes off again - spring out of bed! AHH! It's so cold!!
6:00 - 6:15 am - leave for Emory, depending on dressing speed
6:45 - 7:10 am - arrive at Emory, depending on traffic and leaving time
7:00 - 8:00 am - drink my coffee, read Bible, answer email, update lab notes
8:00 - 9:00 am - study virology
9:00 - 10:00 am - study virology or start experiments etc
10:00 - 6:00 pm - STUFF during the day
6:00 - 6:30 pm - leave Emory
7:30 pm - arrive at home
7:30 - 8:30 pm - eat dinner, talk to parents / John
8:30 - 9:00 pm - take shower
9:00 pm - go to bed
Until next time!
1.27.15, Tuesday, How can I go faster? My favorite question.
I have a certain speed that I consider "acceptable" and right now, that speed is not happening. Sometimes, doing research feels like being a car stuck in mud revving the engine. When that happens to me, it's because I don't know how to do the next step and am trying to figure it out. Like now.
It's very hard to not throw everything else down the toilet - studying virology, eating, even teaching Sam - and just glue my face to my computer screen and try to get the next experiment up and running. It's like crack. Gotta do it. GO GO GO!
But this morning, I studied some virology. Then I found out - oh crap! - TODAY was lab meeting! It just got moved to today last week. I also found out that the construction workers were replacing two of the doors to our lab. I had no idea. Josh had been told at first that it would take 2 weeks. Instead, we found out shortly after he told me this, that no, it was 2 DAYS. Thank goodness. I was worried about dust in the cell culture room, but they did put up plastic and tape it around the door frame to keep such out, thank goodness.
After lab meeting, I went over with Sam about how to run his protein gel. It was his first gel! And he did it largely all by himself! I had to go to class - I felt terrible abandoning him - but he said he was not upset. And you know - he did great. I'm so proud.
Discussing and asking some questions to Becky (Conticello lab), I figured out how to sequence my plasmid. We send our samples to Genewiz. But I had questions about what primers I should pick, etc. Becky is so sweet and she helps me with the simplest questions. I don't do a lot of sequencing or cloning and she does a ton of both. I'm sequencing my plasmids 1) to make sure they are what they should be - just because 2) mostly #1, and also, to have a basis for my records and to shuttle the insert into a new vector.
Tomorrow I shall send in the samples and PLAN CLONING THINGS YESSSSS!! HAHA!! Til next time. :)
1.26.15, Monday, cool seminars about nanoparticles and antimicrobial plastic and reading about how much mammalian protein expression sucks (beware, technical-ish)
Evening - seminar by Jason Locklin (UGA): Khalid hosted Jason Locklin, a surface chemist, to give a seminar this evening - which was really interesting. He talked about what the active ingredient of lysol was and how he had essentially attached a derivative of the same molecule to a kind of other molecule group that would react with plastic (using radical chemistry). He was able to spray this mixture onto plastics and make them antimicrobial (see here and here)! He showed that it would inhibit mold on shower curtains and ocean nets used for farming fish. He's in the process of patenting it right now and lots of people have approached him to use it on their stuff. It's such simple chemistry that he did, he said, that he was embarrassed to talk about it for a while. But it won't make socks permanently not smell, as some news agencies went wild and claimed. Aw man - I'm too tired to write all the details. He discussed a lot.
Future plans: After seminar, I talked to Sam Hong a long time about mammalian expression and cloning (he's in biochem dept). I decided that I'm going to try to express my protein in E.coli first, before I try the AWFUL mammalian cell route. I think it can work in mammalian cells. But, why not try the easy way first? There's some other reasons I came to this conclusion, but I'm too tired now to write them. Until tomorrow!
1.25.15 Sunday, thoughts about last week and look ahead
My current student is awesome! Last week was crazy hectic, but we survived! I have a *very* good senior undergrad student right now - Sam Druzak - formerly from Oxford College. We were both trained by Ms. Harmon and have very similar mindsets. He's fast becoming an excellent clone of me and I cannot wait to see what he can pull off this spring and for a few months this summer! Sam has spoiled me - I do not expect students that I get to be so good - I really lucked out.
Activities last week: On Monday, Sam and I made chemically competent cells (cells that can take up DNA upon heat shock). On Wednesday, I toured the week's speaker, Joe Chihade, who spoke about unusual alanine tRNA synthetases - which were a lot more cool and interesting than I expected! On Friday, I interviewed a homeschool student for this summer and toured him through our lab. It was really a crazy week but I had fun!
To work on this next week: I do need to spend more time for my virology class though and working on learning my new experiment set, to express protein in mammalian cells. I'm also hoping to help Sam get to his activity assays on his protein - once he knows how to do those experiments - he'll have a whole new world to explore.
More updates and photos soon! I do not promise that these statuses will make sense - when I write things quickly, they will tend to be more technical. Elsewhere, I try to avoid that. :D Love you guys!
UPDATE (9:37 pm): I wanted to write more thoughts about the possible publication I found out about - that was a huge shocker this past week - but I'm not going to get time. Maybe next week / weekend! This week is going to be nuts, but it'll be fun too! Thank you for your patience as I try to balance so many things - I'm trying to get to everyone who has emailed me in a timely manner. I'll try to keep updating the blog throughout the week so you can know what we're up to. You guys are awesome!
1.5.15 Monday, protein expression, Styrofoam patty and Kevin
Styrofoam patty: Check out the Styrofoam patty I made (by accident)! I melted about 20 Styrofoam cups and left them in a beaker of acetone that was parafilmed. Someone poked a hole in the parafilm, so the acetone had all evaporated and left a hard crusty patty of plastic. The top was hard, but the bottom was like a clear gel. As soon as I took it out, it started turning white. It was pretty neat.