Tag Archives: RF2016-Week4

The typical day in the Pickup lab begins at around 10:00 AM. I usually check in with Dr. Pickup and our lab tech Nicole to see how they’re doing and make sure I know what to plan to work on for the day.

Every other day we split our cell lines and transfer them to new flasks so that they aren’t too confluent; if that is on the list for the day, then I’ll bring our cart of supplies to the tissue culture room on the floor and work with the cells. Otherwise, I typically will run experiments with  DNA (minipreps, ethanol precipitations, restriction enzyme digests, gel electrophoresis, etc). Usually these experiments have some wait-time associated with them as they run to completion, so in between I tend to read papers, plan future experiments to run, or eat lunch. I’ll also check back in with Dr. Pickup to make sure I’m on the right track and to get more insight into the details of the project and experiments I’m involved with.

Each day in the Pickup lab is enjoyable and is typically complete by about 6:00, so if an experiment can’t be left overnight we tend to plan it to be finished by then. It’s very nice to be in a smaller lab, in that I have the opportunity to speak with Dr. Pickup daily about his work and my project. It’s fantastic to get that insight frequently and has been very valuable to my research experience.

My day in the lab begins at around 10 am. As I mentioned in my week one blog post, my day typically revolves around trial and error. Although I am getting much better at using MATLAB, I continue to refer to online documentation, or textbooks that my secondary mentor provided to me, in order to properly use commands.

After starting up MATLAB, I usually just pick up where I left off the day before. I always have my lab notebook present in case I need to organize my thoughts, or plan my approach. Another technique I use – and recommend – is to think about code in the most rudimentary steps. Although computers are extremely sophisticated machines, they cannot predict our thoughts. I’m constantly thinking to myself, “ I need to take that variable from over there, manipulate it, and place it over here.” This thought process has really helped me understand how MATLAB, and code, works.

Whenever I complete a small segment of my project, I touch base with my secondary mentor to make sure I am going on the right track. I also speak to him whenever I think of another possible method to accomplish a desired goal. Although I am working with code, I still try to challenge myself by thinking of questions or different methods to achieve a task. It’s also important for me to never lose sight of the end goal, whenever I feel sidetracked or stuck with a segment of code. I leave lab anywhere between 5 pm and 7 pm, and I always make a note to myself in my lab notebook about where I left off.

No day is the same working in the Poss Lab. Every day consists of new experiments to carry out, new things to learn, and new questions to ask.

The first two weeks of lab largely consisted of shadowing my secondary mentor and learning basic protocols. After gaining experience and comfort in conducting PCR reactions, gel electrophoresis, heart extractions, embryo injections, cloning, and cryosectioning (just to name a few), I have really enjoyed gaining independence in my work.

My project looks at 4 different genes and they’re currently all at different stages in my experiments. I have already ran in situ hybridization for one gene while I’m still at step 1 in trying to isolate the segment of DNA I want for another gene. Thus, every day I continue working on my project, whether it’s the final step for transcribing my RNA probe or trying to troubleshoot a PCR for the tenth time. My days will usually involve running multiple PCR reactions and gels. If my gel runs properly, I can extract the DNA band and purify it. Then, I clone the DNA into a plasmid and transform it in bacteria. After the bacteria grows, I set up a colony PCR to verify the DNA segment and send the plasmid for DNA sequencing. If the DNA sequence looks good, I inoculate more bacteria, extract the DNA with a midi-prep, linearize the plasmid, purify, and transcribe (if all goes smoothly) with many PCR’s and gels in between in order to double check each step. Usually, a step won’t work correctly and I have to try to figure out what the problem is. Troubleshooting a step may take days, so I repeat a step many times, changing one thing at a time in order to determine what is wrong.

Depending on the day, I may also practice embryo injections which involve setting up zebrafish matings the day before. I collect the embryos, prepare the injection mixture, and inject hundreds of zebrafish embryos. I may also need to make cDNA for my PCR which involves dissecting zebrafish hearts, extracting the RNA, and reverse transcribing it to make cDNA. I am usually conducting many experiments at once. While I’m waiting for my PCR, I may be embedding hearts in tissue freezing medium or screening zebrafish for mutants. Some days I am busy going back and forth from the lab to the fish room. While other days I have more waiting time when I can read some papers.

My routine largely consists of the same protocols, but working every day in lab is still exciting. Although creating an RNA probe for each gene involves the same steps, each gene behaves differently. Thus, applying each step to each gene creates different results, so I never know what I’m going to get. Sometimes, things don’t work out so it can be challenging but I enjoy trying to solve the puzzle. I have definitely learned that science takes time but I am excited to discover what it will reveal.

I don’t have a typical day but I do have a typical week in Kuo Lab. I conduct a thorough mutagenesis experiment where I complete multiple different steps and ultimately mutate the desired sites of the gene.

I start with preparing agar plates (plates that have the necessary conditions for bacteria to breed) and store them in the cold room (4 °C) for later use. Then I prepare my PCR samples by aliquoting the necessary reagents and template DNA plasmid. I add primers – specifically designed to mutate the desired codons of the amino acids – and run the PCR samples in a thermocycler where they get amplified. After that, I run a gel electrophoresis to check whether the reaction actually worked. Having confirmed that PCRs worked just fine, I use kinase, ligase and Dpn1 enzymes to convert the linear DNA product to circular (because bacteria would chop up the newly transformed DNA if it were linear) and get rid of the original non-mutated  templates. Now our products are ready for transformation. I start transforming the mutated DNA plasmid into bacteria to further increase the number of the plasmids. I thaw the frozen bacteria and aliquot the PCR products into the bacteria tubes. I incubate the bacteria in ice for 30 minutes and then do a heat shock by placing the tubes into the heat block at 42 °C for 30 seconds. This helps transformation since the pores in bacteria enlarge and take up the plasmid DNA more easily. After this step, I place the bacteria to agar plates and do an overnight culture to grow bacteria colonies.

Next day, I look at my plates first thing in the morning. If some of the plates don’t have any colonies, I start over for them and redo the previous steps (I know, kind of frustrating). For the ones that worked, I pick three colonies from each plate and prepare them for plasmid DNA purification by doing an overnight liquid culture. The next day I do a miniprep to purify the mutated plasmid DNA. I add buffers and centrifuge the cells multiple times in order to digest them and precipitate the unwanted biomolecules (fats, carbohydrates, proteins, RNA and bacterial DNA). After that I use a spectrophotometer to measure the concentration of the DNA. To make sure that my plasmid DNAs have the mutated gene insert, I use restriction enzymes and run another gel electrophoresis. After confirming, I send my samples for sequencing and get the results the next day.

Of course, none of this process goes smoothly. When I started my project the first week, I thought I would be done with this part of the project (generating mutated plasmid DNA) in two weeks. I can now see how naive I was. During these steps, we had to readjust the temperatures for thermocyclers multiple times to get a better yield from PCR and ensure transformation. We had to add new chemicals to PCRs because some of our primers had rich G-C nucleotide contents which made them bind to the other strand very strongly and disrupted the PCR. Sometimes everything seemed to work and we sent the plasmids for sequencing, only to find out that the original template DNA was transformed not the mutated ones. So, we had to restart everything and wait for Dpn1 enzyme to operate much longer to thoroughly get rid of the original template DNA. I will start my fifth week tomorrow and I still need two more mutations to proceed to the final part of my project. Although there were times I got really frustrated about my experiments, these experiences have taught me that troubleshooting is very important in scientific process and we have to be very resilient to reach success. I am pretty sure that my Western Blots won’t go as planned either and I’ll have to perform a lot of them. But I believe I will be able to overcome the hardships I encounter by trying new adjustments and not giving up until I succeed.

My daily activities in lab really depend on what point of the experiment we’re at. Since my project is such a giant experiment, it takes up most of my time during the day, though I do help work on another smaller experiment that’s also going on. I generally arrive in lab around 10 AM. The more intense days have me working continuously from 10 to 5, while on the easier days I have time to read papers and work on some smaller assignments (like blog posts or the upcoming chalk talk).

The first few weeks at lab were pretty intense, since we all had to work together to prepare the experiment due to the sheer quantity of plates (2,304) needed. In the mornings, I would cover an entire lab bench in petri dishes (it turns out your standard lab bench can fit exactly 450 3 millimeter petri dishes on it). Then we needed to prepare the agar, which can fortunately be done on a larger scale using the autoclave machine down the hall in the Biological Sciences building. This way, we can heat up around 12 bottles of agar at once in about an hour. After filling up all the plates, they needed to be chilled overnight so that no mold or fungus grew in the agar. Making all of the plates basically took up the entire first week.

The second week, we had to put 20 Arabidopsis thaliana seeds in each plate. The maternal plants for this project were grown before the program started, but a bunch of plants of each genotype were grown, and then placed in their respective simulated environments. Each of the plants had to be wrapped in a plastic tube in order to prevent cross-pollination. Then, at maturity, the seeds of each plant were collected into tubes, which I then placed into their respective plates. Since the seeds are extremely small (not much more than a speck), I had to use a thin probe and place them one by one on the agar. Then, trays with filters were filled with 36 plates in order to simulate a certain light quality environment. After being filled, we had to take them to temperature-controlled chambers that are either down the hall in BioSci, or in the phytotron (a rather dark and scary place filled with dozens of tall, humming, machines) in French Science.

I was able to start doing germination censuses for the project in week 3. This involves taking the trays out of their chamber, examining the plates under a microscope, and counting the number of seeds that have germinated (which is when you can see the radicle poking out of the seed coat). If all of the seeds are fully germinated, then they can be thrown in a bin to be autoclaved (which kills all of the cells so that they don’t cross-pollinate with natural genotypes!). Then the trays go back in the chambers. The whole process is usually finished by lunch, especially since we have 3-4 people helping out at once. The rest of my day is spent getting caught up on papers or taking care of other plants down in the phytotron. By next week’s census, I should have enough data to begin analyzing the results!