...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...

... 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...

...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...

...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...

...interest, it may be time to look at the indirect approach - which means it’s time to talk about secondary...technique, and sometimes each individual assay, will need to be developed and optimized for the samples...that a trusted stock of secondary antibodies saves time and money when developing assays.  Protocol architecture....  Even though the secondary antibody incubation time is often shorter than that of the primary antibody... to take place over multiple days. Increased run time is considered one of the major drawbacks of the ...attaching to a single primary antibody, it is sometimes assumed that the direct method is more quantitative... the direct or the indirect method.  New Methods Time is a precious commodity in the lab and eliminating...

...proteins for live cell imaging can be excited many times, thereby producing a large number of emitted photons...number of excitation/emission cycles per unit of time, decreasing the likelihood that a fluorophore will...are excited with very intense light (for a brief time), whereas in widefield imaging fluorophores are ...with relatively low light levels (for a prolonged time). Other illumination strategies, such as 2-photon-excitation...performed that measures fluorescence intensity over time. In order to predict how fluorophores perform in...in ‘real experiments’, it is advisable to perform time-lapse imaging of cells producing the fluorescent...comparing how the fluorescence intensity changes over time, the photostability of the fluorescent proteins ...

... five rules. By 2020, they had used this data-optimized assembly design (DAD) to build out three tools...you. Once you input a sequence, SplitSet finds optimal fusion sites to split it into multiple fragments...Table 1).   Enzyme Temperature Incubation time/cycling Overhang length T4 DNA ligase 25...incredibly complex reaction possible, NEB also optimized the thermocycler protocols and made sure that ...complex reactions benefited from longer incubation times and didn’t necessarily require cycling. The Pros...assembly types, circular assemblies yielded 500 times the number of plaques than the linear ones. The ...selection process and benefited from a longer reaction time of 15 hours. This suggests that 52 fragments may...

... utility in the least amount of time. Though I now spend far less time answering help emails and phone...call. Though my experience is based solely on my time at Addgene, I’m confident that these tips and tricks... call is a big commitment when you’re busy. Your time is precious. Here are a few things to consider before...with your product - this could save you a ton of time. I've definitely thrown out all the paperwork associated...know was just buried beneath someone's lunch. Many times, I’m pleasantly surprised to discover that the most...We understand how exasperating it can be to lose time and money on a potentially faulty product. Please...response, however, consider following up. Ask for a timeline if you are not given one. Though an initial response...

...ahead of time!). This notice can be shortened as the meeting settles in and people get the time recurrently...bench I certainly had concerns that I may not have time to stay current or enjoy that discussion. Luckily...specific field(s). Most anyone who has spent any time in academia has likely had the opportunity to attend...that occurred once a week for several months at a time during the spring and fall semesters. Our scheduling...for the first meeting so that people can set aside time in their schedules and read the paper (PS: All participants...successful meeting: a clear purpose and effective time management, for starters.  Now, you are ready for... for scheduling conflicts. This helped us find a time that accommodates more people. The feedback could...

...contribute to my community. This past year, I spent some time thinking about how I could combine all of these ...event-planning base and will know how to reserve space, time events, and network for volunteers. Speaking of...this opportunity as coming from networking at the time, but that means it was the best kind of networking... a presentation for 8th graders was a little intimidating. Too simplistic and I would insult the students...does, and how one becomes a scientist. I gave a timeline of my science life, starting back to Mr. Kelley...communicators.  What can CRISPR do? I spent more time on this section of the presentation because, in ...Fig. 2). One thing I will likely change for next time is giving fewer examples and think of a way to make...

...and grant applications in real-time so they can provide input at the times that work best for them. This...challenges can be exaggerated when communicating across time zones, digitally, and asynchronously. The best way...collaboration includes scientists working in different time zones, it’s important that everyone can access data...communication of emailing documents to one person at a time. Just be sure there is a point person responsible... collaboration. Some things to figure out are timelines for experiments and manuscripts, who is responsible...reaching out if any milestones are missed. Spend time in person Presenting the results from my lab...circumstances permit, think about having collaborators spend time with each other in person. One possibility is to...

...Out of the genome Tips for a first time CRISPR User (by a first time CRISPR User) Additional resources...known. If a strong candidate emerges, downstream optimization is performed to take the drug to the pre-clinical...and can help weed out a lot of poor drugs before time and money gets put into them in those later stages...to inhibit, activate, or knockout many genes at a time to identify ‘hits’ - genes that affect disease progression...onset. These hits can reveal individual genes or sometimes even genetic pathways that are worth investigating...toxicity. This added QC is where CRISPR can save time and money to make sure quality candidates are selected...also been added to the CRISPR technology to allow time-controlled edits to occur, such as in the context...

...been requested 20+ times   A red flame means a plasmid has been requested 50+ times   A blue flame means...deposited plasmids have been requested more than 100 times each, thereby achieving the coveted “blue flame”...influential that it is requested from Addgene many times as the technology disseminates throughout the research...means a plasmid has been requested 100+ times Since we so sincerely appreciate our depositor’s dedication...Addgene that has been distributed more than 100 times. We hope these desktop trophies will serve as badges...can imagine, answering plasmid requests can be a time-consuming chore for labs that have popular plasmids...material transfer agreements, thereby freeing up time for our depositors to continue conducting research...

...culture for an extended period of time (>10 passages or >3 months). Other times cell lines are unavailable from...concern for the discipline (or lack thereof) in times gone past but these days it’s never been easier ...biology. Engaging in this practice would seem to save time and money but cells lose provenance (this is the...closely followed by T24 bladder cancer cells. Estimates of the amount of money wasted in basic oncological...case if you obtain a cell line from a colleague. Time and money are wasted, papers are retracted and there...cells from a repository like the ATCC, there are times when it is important to check their identity. For...once you’ve passaged your current batch too many times or you suspect there is contamination. You would...