July 13th - 24th, 2015, My RNA has died a lot
In summary, these two weeks, I attempted many labelings of RNA all of which degraded. I am now trying to find the source of the contamination. ARG! RNA! Ha ha, but if it wasn't challenging, what fun would it be?
July 12, 2015, Sunday, tired, making to-do lists and hanging out with John
Today, I just went to church, talked to some people, and came up here to Gigabites Cafe to the Warmachine tournament to hang out with John. I've been consolidating my work load in lists. I ironed John's shirts before coming per the usual. I think on Monday, I will spend it catching up and starting new RNA labeling so I can run some tests I need to run. I already have two experiments in mind for Grace. We'll see if I can get some stuff done, but I think Monday is going to be my catch up day. The weekend has unfortunately been exhausting and not really fruitful toward getting my work done. But, I'm very peaceful, I enjoyed it - things are good - I have no complaints. :) Just tired.
July 11, 2015, Saturday, running lots of errands, lots of errands
I was busy. Saturday, I collected presents for lots of people - my mom, whose birthday is next Tuesday - and for Weiwei, because he's leaving us in August to be a professor in NY and such. I went to grocery store and stocked up on these fruit drinks I found that I like, as well as buying necessities. Went to Hobby lobby to get decorations for mom's birthday. Mostly, I did all the things that I didn't have time to do during the week and it wore me out. It was also really hot.
John plays in Warmachine tournament. Meanwhile, John went to an all-day tournament for Warmachine in Marietta. I drove up there after my errands and a nap. He bought me a brownie Sunday from Mazzy's here they ate, which was delicious. But come 8:15 pm I had to go back home because I was so tired. We ended up going to be quite late. I just read until he got home. John won his first two games, but played Brian White in the third round and lost. This is acceptable though because Brian is a world-champion Warmachine player. However, even so, he was one of eight people who made it into the second round on Sunday.
John plays in Warmachine tournament. Meanwhile, John went to an all-day tournament for Warmachine in Marietta. I drove up there after my errands and a nap. He bought me a brownie Sunday from Mazzy's here they ate, which was delicious. But come 8:15 pm I had to go back home because I was so tired. We ended up going to be quite late. I just read until he got home. John won his first two games, but played Brian White in the third round and lost. This is acceptable though because Brian is a world-champion Warmachine player. However, even so, he was one of eight people who made it into the second round on Sunday.
July 10, 2015, Friday, running two gels
Grace's data is cool and Erin defended her thesis! I got to show Khalid Grace's data and he thought it was really interesting as well. Erin (of Dyer lab and Kevin's girlfriend) defended her thesis at 1:30 p.m.! I wished I had gone, but I just found out that morning and planned experiments which I had wanted to do, so I did those instead. She passed though and it was awesome I heard!
Experiments are slow but accomplished! My experiments took way longer than I expected to setup, because while aliquotting out the reactions, I ran out of both of my small stocks of RNA strands, so had to make fresh and Nanodrop them - never a fun prospect. Finally, I got done around 8 p.m. Also, I left my phone at home by accident, which worried John. But I sent him email updates, which worked.
Experiments are slow but accomplished! My experiments took way longer than I expected to setup, because while aliquotting out the reactions, I ran out of both of my small stocks of RNA strands, so had to make fresh and Nanodrop them - never a fun prospect. Finally, I got done around 8 p.m. Also, I left my phone at home by accident, which worried John. But I sent him email updates, which worked.
July 9, 2015, Thursday, freezing protein
I finished concentrating my protein and froze it. Today, I was worn out, but I ended up freezing all my protein into smaller aliquots. However, it was not quite concentrated enough when I checked it in the morning, so I allowed it to dry three hours at room temp. Made me nervous for it though, even though my protein is so robust I wasn't that concerned.
Grace did a pH experiment. Meanwhile, Grace ran a gel of two different pH's to test how our enzyme worked in them both. She did a great job - it showed some pretty cool info! I was impressed. I wasn't sure what would happen with that one.
Grace did a pH experiment. Meanwhile, Grace ran a gel of two different pH's to test how our enzyme worked in them both. She did a great job - it showed some pretty cool info! I was impressed. I wasn't sure what would happen with that one.
July 8, 2015, Wednesday, purifying protein
On Wednesday, Grace worked on practicing pouring an SDS-PAGE gel and loading samples while I isolated my enzyme. I tried a new trick to concentrate it that the Lutz lab told me about, leaving it overnight in the cold room outside of buffer in the dialysis bag to slowly evaporate. I was quite tired this day for some reason and the day was long, but it was really good too.
July 7, 2015, Tuesday, really busy! lab meeting presentation + gels
SO BUSY. Tuesday was insane! LOL After lab meeting, in which I presented, Grace and I briefly discussed a paper on gels that I gave her to read, then launched into doing experiments. We managed to run two gels - I ran one and she ran one. It was a very busy day. Her gel turned out quite nice too!
July 6, 2015, Monday, feeling frustrated
Why I was frustrated. I present in group meeting tomorrow, giving an update on my research. I wanted to get one last gel to show some interesting data in it, but alas, no banana! Then, I was super frustrated, because I didn't know why the gel hadn't worked very well. I was SO upset, I emailed someone in the Raines lab about it who works on RtcB. But, after talking to Khalid and reviewing my old data, a theory / hypothesis about the problem came out, which is a relief. I'm going to be reviewing my stuff for tomorrow so not much time to write tonight.
I came home and jogged -- now it's pouring rain. Looking forward, in some ways, to presenting tomorrow. Then, finishing up protein expression, and untangling this unruly gel.
I came home and jogged -- now it's pouring rain. Looking forward, in some ways, to presenting tomorrow. Then, finishing up protein expression, and untangling this unruly gel.
July 5, 2015, Sunday, Preparing for next week, excited about experiments to come
Today, we were intending to hang out at Brian's house, but ended up staying home. We're watching the Women's Soccer World Cup final: USA vs Japan rematch -- very exciting! I'll probably watch it with half an eye while reading science papers. We went to Golden Corral with John's parents for lunch. I've already ironed all our shirts for next week. I looked into organizing material for a dps set for my second sage and my gunslinger on SWTOR MMO. Talked to an old online friend. Then, after I finish updating these posts, will be working on fixing up a few slides for my presentation on Tuesday, and reading science articles.
Thus is today! More next week! I'm very excited about the experiments to come!
Thus is today! More next week! I'm very excited about the experiments to come!
July 4, 2015, Saturday, Happy July 4th!!
Summary. On July 4th, I pretty much spent the whole day working on our lab's website, then John and I, mom, dad, James and his girlfriend Elizabeth hung out around 8 pm at Snellville, we listened to a band play Cuma Matata, got shaved ice, I read the first 3 chapters of the first book in the Dresden Files (John is reading them) and we popped firecrackers, lifted off a glow globe and watched the fireworks. Then (somehow) we managed to get home in 30 min, beating the traffic, because of the terrific spot Dad found, and watched Independence Day (1996) until 1 a.m. It was AWEsome! I had a lot of fun. For photos of the stuff we did in the evening, see dad and my photos listed here, in family blog, which has the same post in words but more photos.
Glow Globe. I was really excited about that glow globe we launched. We saw them last year, and we really wanted to try them out. So, this year, we launched our own!
Big fireworks are scary. Also, the big fireworks we launched were scary as crap. HOLY COW! They were really loud. After my brother launched about seven of them, I'd had enough. They were moderately terrifying. It'll take a while to get used to those. Our neighbors were also launching their own gigantic ones, some of which were bigger than ours. We got quite the show!
Glow Globe. I was really excited about that glow globe we launched. We saw them last year, and we really wanted to try them out. So, this year, we launched our own!
Big fireworks are scary. Also, the big fireworks we launched were scary as crap. HOLY COW! They were really loud. After my brother launched about seven of them, I'd had enough. They were moderately terrifying. It'll take a while to get used to those. Our neighbors were also launching their own gigantic ones, some of which were bigger than ours. We got quite the show!
July 3, 2015, Friday, John comes to work with me!!
Friday was great! My summer student Grace came in to work. John came with me to work and put together Warmachine models on my desk. John helped out James's girlfriend change the battery in her car while I was putting together an experiment. It rained in torrents randomly throughout the day. All of us -- James, Elizabeth, John, Grace and I -- ate at Slice and Pint too! Before it opened, John, Grace and I went to the cookie store -- and I bought and M&M cookie, John a snickerdoodle cookie, and I got a frosting shooter and cake in a jar. Then, we went to Barnes and Nobles and I bought two Star Wars books.
We did an experiment when we got back, but sadly, it kind of bombed. I'm not exactly sure why, but I'm going to give it another shot Monday. When we came home, for some reason, I was really tired and took a nap until 7 p.m., when dinner was ready.
Having John there all day was such a treat! I loved having him at work. I showed him the -80C freezer and everything, and a few things I was doing with experiments.
We did an experiment when we got back, but sadly, it kind of bombed. I'm not exactly sure why, but I'm going to give it another shot Monday. When we came home, for some reason, I was really tired and took a nap until 7 p.m., when dinner was ready.
Having John there all day was such a treat! I loved having him at work. I showed him the -80C freezer and everything, and a few things I was doing with experiments.
July 2, 2015, Thursday, Protein purification insanity post-poned!
I came to work today all prepared and revved up to work until midnight, and ran into Ben from the Weinert lab. I told him what I was doing and he said in effect, "You know, you could do [this] instead and not do that." And I was like ... *jaw drop* DUH! Obviously. I should have thought of that. I don't have to do the expression and purification in one day after all! So, I ended up deciding to follow his sage advice and not do insanity instead. It was a relief.
What I did instead: Expression and RNA labeling. So I labeled some RNA for an assay later. And I expressed my enzyme (half of what I had planned) but did not purify it. I froze it for later. I just need to do the purification and dialysis in one day. I can split it up at the expression.
Peaceful awesomeness. It was a very peaceful, fun and awesome day -- one of those days that's totally normal, and one is doing one's work, feeling happy and vaguely excited and satisfied with life. I got lots of packages from the stockroom, re-read a Shuman paper and made notes, wrote up Dr. Weinert's and the Lutz lab people's thoughts, wrote my own notes, enjoy a lunch of perlo and bread ... ahh. This is the life. I love no class and just doing what I love best: research and thinking. Thinking and research.
Tomorrow will be better! Tomorrow will be even more awesome because John is off work, so he's going to hang out with me at Emory! I'm so happy about that!!!! Ha ha. What a great week. Until next time!
What I did instead: Expression and RNA labeling. So I labeled some RNA for an assay later. And I expressed my enzyme (half of what I had planned) but did not purify it. I froze it for later. I just need to do the purification and dialysis in one day. I can split it up at the expression.
Peaceful awesomeness. It was a very peaceful, fun and awesome day -- one of those days that's totally normal, and one is doing one's work, feeling happy and vaguely excited and satisfied with life. I got lots of packages from the stockroom, re-read a Shuman paper and made notes, wrote up Dr. Weinert's and the Lutz lab people's thoughts, wrote my own notes, enjoy a lunch of perlo and bread ... ahh. This is the life. I love no class and just doing what I love best: research and thinking. Thinking and research.
Tomorrow will be better! Tomorrow will be even more awesome because John is off work, so he's going to hang out with me at Emory! I'm so happy about that!!!! Ha ha. What a great week. Until next time!
July 1, 2015, Wednesday, Preparing for tomorrow's day of insanity!
Today was busy! But fun! And tomorrow will be ... INSANE. Here's the story.
I drank my coffee in a Yoda glass and it was awesome, oh yeeeeaaahhhh.
Meeting with Dr. Weinert to talk about her centrifuge. Firstly, I met with Dr. Weinert at 9 am for an equipment refresher. I was worried, because last week I made another mistake with her centrifuge - greasing the o-rings with the wrong type of grease - even after I'd written an extensive list of instructions to myself. But she wasn't really upset. I marked off some more things to remember when using it. It's very important, because her ultracentrifuge (that's what it's really called) spins at 120,000 g's and puts the sample tubes under vacuum so the pressure of the g-forces doesn't crush the tubes. It's serious!
Talking to the Lutz lab about protein expression. I talked to Pravine and then Samantha (Sam) a long time about protein expression. My yield was a little low when I expressed it with Grace, so I hashed out with them some ideas of how to increase it and how to concentrate it. I had a lot of fun talking to them and they had some great ideas.
Synthesizing more gold nanoparticles! Today, I synthesized 100 ml gold nanoparticles for the third time. This time, I tried to do all the tricks I'd learned the first two times. Kevin wasn't even here, so I did this one totally by myself! Yun and her undergrad Nick watched. I really hope it worked this time - no fused particles AND most of them the same size - that's what I want.
Preparing for protein expression. Tomorrow, I'm going to express protein and purify it in the same day - the experiment of DOOM of DOOM!! This is one I really am glad I only have to do it once a year. It takes from about 7 am - midnight. I have to do it like this to try out the tips Pravine (Lutz lab) mentioned to me. I'm going to try to concentrate my protein in a unique way -- I think it will work, based upon the tests I ran on it this past week. I'm REALLY EXCITED about it! Maybe I can finally get concentrated protein this way!
Technical jargon - what am I doing to concentrate it:
Talking to the Lutz lab about protein expression. I talked to Pravine and then Samantha (Sam) a long time about protein expression. My yield was a little low when I expressed it with Grace, so I hashed out with them some ideas of how to increase it and how to concentrate it. I had a lot of fun talking to them and they had some great ideas.
Synthesizing more gold nanoparticles! Today, I synthesized 100 ml gold nanoparticles for the third time. This time, I tried to do all the tricks I'd learned the first two times. Kevin wasn't even here, so I did this one totally by myself! Yun and her undergrad Nick watched. I really hope it worked this time - no fused particles AND most of them the same size - that's what I want.
Preparing for protein expression. Tomorrow, I'm going to express protein and purify it in the same day - the experiment of DOOM of DOOM!! This is one I really am glad I only have to do it once a year. It takes from about 7 am - midnight. I have to do it like this to try out the tips Pravine (Lutz lab) mentioned to me. I'm going to try to concentrate my protein in a unique way -- I think it will work, based upon the tests I ran on it this past week. I'm REALLY EXCITED about it! Maybe I can finally get concentrated protein this way!
Technical jargon - what am I doing to concentrate it:
My protein precipitates in Amicon filters when I do buffer exchange on the 7 ml of ~.2-1 mg / ml protein I get. So, I typically dialyze it overnight and freeze the resulting batch as is. Pravine said I could leave the dialysis bag in open air to let the extra water evaporate slowly (keep it in cold room though). That's a genius idea. I think it will work, because I tried to concentrate my protein in the speed vac, vacuuming off the excess liquid, and it worked! The protein concentration got up to 2.1-ish mg / ml! Normally, it would precipitate out. I'm wondering if it worked because the salt and buffer concentrations increased as well, since I'm just removing water, helping the enzyme stay happy. Therefore, this method should work too. But, I wanted to dialyze the protein, then leave it overnight to dry -- I don't want to risk it overnight and not flash frozen any longer than I have to -- thus everything has to be done in one day. To achieve that, I have to do the one day expression / purification insanity. But I think it'll be worth it!!
Figuring out more problems with transcription. I transcribed two small RNAs recently -- one transcribed really well and one did not at all. I think I figured out why the one didn't work. I'm pretty sure it's a sequence-specific issue related to what nucleotides T7 enzyme likes at the beginning of the transcript.
Visiting James. While going home, I saw James driving up to visit his girlfriend Elizabeth (she lives right next to Atwood chemistry). So I visited with them 30 min and met her dog Milo. That was really hilarious!
And tomorrow is a NEW DAY full of more AWESOME! Until next time! Same bat time! Same bat channel!
Pictures of gold nanoparticle synthesis!!
Visiting James. While going home, I saw James driving up to visit his girlfriend Elizabeth (she lives right next to Atwood chemistry). So I visited with them 30 min and met her dog Milo. That was really hilarious!
And tomorrow is a NEW DAY full of more AWESOME! Until next time! Same bat time! Same bat channel!
Pictures of gold nanoparticle synthesis!!