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...reaction, and the timing and number of harvests will all impact the titer. Importantly, optimized conditions ...initial optimization; cell number, media volume, and plasmid concentrations will need to be optimized prior...Additional plasmids will also affect experiment optimization. Before beginning an experiment, it is recommended...additional plasmid for transfection can make these optimization experiments a bit more challenging. With these..., for your experiment. To improve titer, first optimize transfection conditions. As mentioned above, plasmid...efficiency. For some transgenes, transfection optimization simply is not enough to get a sufficient titer...glycol (PEG) precipitation. All of these methods ultimately isolate the viral particles allowing you to concentrate...

...discussion? Or maybe the one that goes way over time so there is no time for audience questions and an audience... negatively affect timing and the liveliness of the discussion (see below on timing). Get good resumes...encourage this. Timing is everything Many of my suggestions are meant to keep the event on time. Nobody likes...on a stage if the room is very big, but for an intimate event, put the panelists on the same level as ... show. The moderator must be very active at all times keep things moving and to prevent any one panelist... to introduce themselves and, voila, 1/2 of your time is gone with no interesting discussion. The audience... part of the publicity. Good panels do not waste time on long introductions. The moderator asks questions...

...maintain fidelity with the stimulus. For example, if you want to stimulate at a fast frequency without...very different responses to long vs. short stimulation times or frequencies (Zhang & Cui, 2015). You’ll...that induce conformational changes with light stimulation to either activate or inhibit a specific receptor...the cell to be excited over a sustained period of time (Gong et al., 2020). Light-gated ion channels are...on and off rapidly with light to control precise timing of the perturbation. Other tools are designed to...cryptochrome 2 (CRY2) (Zhang & Cui, 2015). When stimulated with light, the endogenous proteins activate ...experiments.  Wavelengths Most vectors have an optimal wavelength to use but can be activated by a range...

...Harvesting timepoints will depend on your specific virus, so this is something you may have to optimize for ...increasingly accessible and are useful for saving time and avoiding potentially costly set-ups. However...in-house. High-titer adenoviruses are difficult and time consuming to produce, so most labs opt to purchase... kb, while lentivirus has a capacity of 8–10 kb. Time Viral production in mammalian cells is relatively...taking your first round of virus, called P1 (or sometimes P0), infecting new Sf9 cells, and getting an amplified...eliminate dead cells and cellular debris. While adding time (about three to four days), this second round provides...like lentivirus. You can even produce a P3 virus! Time Sf9 cells will start producing virus within a day...

... editing for a few months. More time is required for its optimization and development before it can truly... "Final Verdict", is that more time is required for NgAgo optimization before we can make any major conclusions...PAM sequence might not always be available at an optimal distance from the target site. For example, NGG...been requested by labs around the globe nearly 400 times. Researchers have been excitedly testing NgAgo's...method and 65% wanted to wait until someone else optimizes an NgAgo protocol for their favorite model system...respondents suggesting that NgAgo can indeed be optimized for genome editing in mammalian cells. However...indicates that it is very tricky to use and fussy to optimize. One reason could be the instability of the supposedly...

...interview was intimidating to me, as I had a hard time thinking about how I could fill this time. I was relieved...spent a lot of time shadowing members of my team to learn what I was to do for my time at Addgene. This...make up the hours I had missed at another time. This made time for me to attend to other commitments, without...The steps of the training process gave me enough time to become familiar with the different types of issues...something called “core hours”. Core hours consist of the time between 10 AM and 3 PM, when most meetings are scheduled...the three hours (outside of the core hours) at a time that works for you. For example, if I needed to ...missing work. As long as my work was finished on time, there were no issues. Another example of helpful...

...purpose and finite goals. For example, when it came time to update how we make and distribute kits, I helped... difficult assay with her eyes closed. How many times have you ever seen a scientific article attributed...meetings and we’ve volunteered our ideas, skills, and time to the cause ever since. Unlike the previous two...The benefits of a team So why is it helpful, or sometimes even necessary, to build a team in the first place... when Thomas Jefferson dined alone.” There was a time in human history when an intelligent, motivated ...diverse group of people to provide that base. Sometimes there’s just too much work If you are one of those...other people who have a stake in the outcome will ultimately improve the quality of the work as well. Social...

... Most of the time, updating these libraries is straight forward. However, there are times where a library...genders, educational, and professional backgrounds. Sometimes this can lead to wildly different ideas of how...blind spots. Blind spots often result in less than optimal user experience and the overall soundness of the...stories, and work on stories during short periods of time called sprints. The team is mindful of the number... decide how to best approach it in our code.  Sometimes while thinking through these steps, we discover...code is written, we are able to use our developers’ time efficiently. While writing the code, we may need...developing new features and fixing bugs: we take time during each sprint to do some maintenance work on...

...lower than optimal drug concentration. Drug resistance is not a black or white issue. Sometimes bacteria...killed by a higher concentration, or sometimes more exposure time is needed for the drug to work… if the...superbug’. Superbugs are often very difficult and very time consuming to treat. Learn How Antibiotic Resistance...naturally resistant but has become resistant somehow overtime. One fantastic example of intrinsic resistance...bacterial populations to antibiotics at unnecessary times, putting pressure on resistant bacteria to over-...When you go to the doctor, it will very likely take time to determine if you have an antibiotic resistant...antibiotic resistance is very likely to develop over time. It isn’t feasible to completely eliminate the development...

...figuring out when to schedule times to write.  Pomodoro technique timer  One time management technique that...made your tables and figures. Now it's time to write. Sometimes, it's hard to get those words out and ...kitchen timer that the technique’s developer used as a university student.   The Pomodoro timer is ...borrow this book from your library or don’t have time to read the book, this list summarizes the key points...schedule, even if you only write for a short amount of time each week, and there’s advice on how to actually...great for generating citations for a few papers at a time. Mendeley Mendeley works much like other popular... Hello PhD Podcast episode 15 "Simple tricks for time management: the pomodoro technique": This podcast...

...You may need to optimize the type of block used, the concentration, or the incubation time depending on ...target molecule. You may need to optimize the concentration and incubation time. Secondary Antibody Unsurprisingly...fluorophore. You may need to optimize the concentration and incubation time for this step as well. Preservation...Generating and validating primary antibodies is a time consuming and expensive process. Consider the vast...Multiplexed IF can also require some additional optimization as each antibody performs differently and some...to the manufacturer’s recommendations.  As you optimize your protocol, always remember to include controls...significant impact on the protocol you use and the ultimate outcome of your assay. So how do you choose good...

...obstacles. Take time to consider the order of your narrative. Scientists do this all the time, you just need... quality slides with sufficient time to prepare (and it does take time). For important talks, ask your...speaking is skill that any scientist can learn.  Any time you give a science talk, you are also giving a job... the occasion.  I speak on the same topics many times, but I always seek out information about the particular...your research or work will write itself when the time comes. 5. Choose interesting content Talk about ...public presentation if you are doing it for the first time? Practice talks also help a speaker determine how...adjust content and slides to better suit the target time frame.   I suggest that all graduate students and...

...short period of time, which would prevent the accumulation of unwanted mutations over time. DMD is a good...editing. In order for the therapy to persist over time, stem cell editing would be highly desirable. Should...non-homologous end joining (NHEJ) pathway. It’s estimated that very little dystrophin correction (about ...could make a huge difference in DMD, and it’s estimated that exon skipping therapies would be applicable...dystrophin-expressing muscle cells increases over time, and all three groups confirm dystrophin expression... still needed. First, CRISPR delivery must be optimized to: 1. Reach a high percentage of muscle cells...immunogenicity of the AAV vector Once delivery has been optimized, it’s important to ascertain how long the rescue...

...efficiency counts! The optimized enzyme can be used with any of the PE2-5 approaches, sometimes referred to as...homology-directed repair Homology-directed repair (HDR) stimulated by double strand breaks has been widely used ...everything — prime editing typically needs to be optimized for each application, and the efficacy varies ...precise and versatile, but it requires careful optimization to achieve the best efficiency. The type of ...efficiency can vary widely — so its inventors kept optimizing it. Using a pooled CRISPRi screen, the Liu lab...strand. Temporarily inhibiting this complex may allow time for 5′ flap exonucleases and DNA ligases to act ...originally introduced, rely on the PE2 enzyme. PEmax: Optimizing prime editor sequence to improve expression and...

...achieve your intended next career stage and create a timeline for achieving these goals. Planning is often more...months depending on the revisions and the amount of time spent shopping a manuscript. Manuscript writing ...available the potential mentor is. If the person makes time for you, as needed, this is a strong indicator that...relatively simple experiment. However, by taking this time, you can help ensure that you understand the methodology...and dirty" experiments often eat far more of your time as they can result in spurious yet intriguing findings...productive results and thereby help decrease the time to your first manuscripts. 4. Balance focus and ...seemingly unrelated problem. However, you have finite time and resources. You typically have 4-5 years to make...

...optogenetics differ are: Timing Targeted manipulation Controlling stimulation Invasiveness  Choosing ...Controlling Stimulation Some studies rely on delivering reversible or varied amounts of stimulation to receptors...activation (chemogenetics) or light (optogenetics).   Timing Optogenetics offers a high degree of temporal resolution...Chemogenetics does not offer the same level of control over timing. Because chemogenetics relies on chemical activation... elicit an immediate response or track the exact time of activation. Receptor activity is also drawn out...injected, this is not as precise and the offset of stimulation is gradual. Invasiveness Both optogenetic and...experimental design.   Optogenetics Chemogenetics Timing Fast activation/deactivation Prolonged activation...

...animal has been exposed to morphine or other stimulating drugs, it develops an enhanced propensity for...Mice with higher IL-1 beta production spent more time in the open arm and were less risk averse. Similar... and a dark (protected) area. When measuring the time spent in a particular part of the apparatus, it ...that the IL-1 beta overexpressing mice spent more time in the risky bright place, driven by their motivation...reflects a state of behavioral despair, and the more time the animal spends in the passive mode, the stronger...forced swim test as seen by increasing the ratio of time spent actively swimming versus passive floating....receptor protein that activates neurons upon light stimulation. When they activated the Drd1 neurons 24 hours...

... to full use! Common conditions to optimize include incubation time, antibiotic conditions, and amount...titering and optimizing before you freeze down your viral preps!)  Once you’ve optimized your transduction...amplification protocols. Rather, each protocol is optimized for a single, specific library.  If you have a...their methods sections to see what worked.  Ready, optimize, transduce!  If you’re ordering a pre-made library... plug-and-play: you’ll still need to titer and optimize before you can transduce your cells.  Titering... scientists to perform functional titering and optimize transduction efficiency. Transduction efficiency...vary across cell types and generally requires optimization of many variables, such as seeding density and...

...need to be optimized for each specific sample type. And, if that weren’t bad enough, optimization of one ... digestion, but is harsher and requires more optimization. Enzymatic digestion, while gentler on the sample... ChIP. If you’re using an antibody for the first time, look for one that has been validated in multiple...used, but from the difficulty of developing an optimized protocol - it’s a bit like Goldilocks and the ...one step can affect the optimization of another. For instance, a sample may need more or less cross-linking...Sonication (or enzymatic digest) will also have to be optimized for both tissue or cell type, amount of protein...length for your output assay. A longer sonication time could mean needing to increase crosslinking to counteract...

...experiment.  For example, I used to constantly underestimate time needed to prep for an experiment (labeling...again, and the same goes for your workflow. Next time you make a mistake, instead of silently vowing never...rushing, no matter how hard I tried to build in extra time before the experiment. What fixed it was not trying...fields, and military bases, checklists are the ultimate tool for doing things accurately. Thankfully, ...You may find that after running a protocol a few times, you no longer need the full checklist. Great! But...doing, and doing well, so make sure you’re taking time to do them carefully and thoroughly. Have a physical... things legible. It is faster to spend more prep time on an experiment than it is to rerun an experiment...