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...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...

...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...

...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...

... so you can rely on nap times, after bedtimes, or before they wake up for time to do your own thing. Build...well as time for breaks to stretch, eat, and rest. Try the Pomodoro Technique or the Flowtime Technique... on. Practice self-care and stay connected Find time for exercise. Many studios and gyms are now offering...social support are even more necessary during these times of stress due to #COVIDー19. Receive and provide ... social media professionally but haven’t had the time until now.  Network. There’s a lot of networking...made for kids. Plan out meals and snacks ahead of time.  Find learning activities for kids. For example...

...irreproducible results to future work cost both time and money with estimates reaching in the billions of dollars ...apply these findings in their own work ultimately wasting both time and money. The obvious solution to ...lab and the entire research enterprise time and funds. It’s time to start putting them into practice!   ...program beneath it. You can jump to the different time stamps in the description to watch any sections ... body works, generating new therapeutics, and sometimes formulating new biological concepts. However, ...the final manuscript regardless of whether the ultimate results were positive or negative. I can feel ...of pre-registration doesn’t come to fruition any time soon, the Center for Open Science does host a platform...

...At the time when we spoke, Susan was helping cover social media while the Hutch hired a full-time social...directions is sometimes at odds with the space needed to be creative. “You need a lot of time to let you ...you mind process things. Sometimes finding that uninterrupted time to be creative is challenging,” says ...written.” Ultimately, Susan feels she was lucky to have been in the right place at the right time when she...research for a general audience. She then got a full time position with a Washington D.C.-based communications...

...Before you begin In the best case, you know ahead of time that you want to probe your blot with multiple antibodies...sample. Pro tip! Because some protein is lost each time you strip a blot, you should not compare the strength...stripping means more sample loss. If you are still optimizing your system, start with a mild stripping solution...5–30 minutes at room temperature. The incubation time will depend on your blot — antibodies that bind ...! βME is a reducing agent and gets oxidized over time, so it should always be added fresh to solutions...washing is critical. Assess the stripping Now it’s time to check to make sure the antibodies from your first...re-incubating with your secondary antibody. This is the one time in western blotting that you’re looking for a “blank...

...residue peptide to the C-terminus of human codon-optimized  cryptochrome 2 (CRY2) from Arabidopsis thaliana..., a researcher can cause them to interact by stimulation with blue light.  Similarly, light can also be... and is reversible upon termination of light stimulation. CRY2clust enables faster assembly and disassembly...integrated into a Ca2+ -modulating optogenetic system (OptoSTIM1), caused two-fold faster changes in intracellular...Listen to the soCoChR podcast segment Optogenetic stimulation is a powerful tool used by neuroscientists to...artifactual PSCs commonly observed with direct stimulation are largely absent in the soCoChR-expressing ... concentrations in several beverages could be estimated by measuring growth of the ΔguaB pDCAF3 strain...

...was a machine that could make your life a hundred times easier. We’ve certainly felt this way at Addgene...month!), manual transformations were taking a lot of time. Enter the Biomek FXP! We wanted a machine that ... the oppotunity to use the FXP to automate and optimize more protocols in the future - something that ...doing about 50 transformations in the same amount of time by hand! Setting up a basic protocol on the Biomek...purify DNA from many bacterial samples at the same time as opposed to preparing each sample individually...developed a protocol to run up to 192 samples at a time. It follows all the same steps and uses the similar...assistant, but with increased plasmid demand it was too time consuming to complete this process by hand and we...

...the TSS but you can worry less about sequence optimality because you generally have fewer sequences to...so many possibilities, picking a gRNA with an optimized sequence is of primary importance (more on this...subsequent screening for successful edits. This is a very time consuming process and should not be undertaken lightly... for this technology, and the user may need to optimize numerous parameters to generate the desired edit...have a dozen or so gRNAs to choose from in the optimal location. It is also important to have good information...approximately equal importance in design – an optimized sequence will do little if it is in the wrong ...there are fewer gRNA to choose from, and thus an optimal sequence may not be available. Predicting gRNA ...

...-- as of June 4, 2020, COVID-19 has caused an estimated total of 380,000 deaths worldwide (statistics ... of plasmids ordered for each paper. Rough estimation of COVID-19 effect on plasmid requests Before...plasmid request activity around March. A rough estimate shows that activity level in March was around ... of the world encountered COVID-19 at different times. Most notably, China was affected by COVID-19 earlier...lockdown. To allow our model to give uncertainty estimates for each parameter, we added log-normal noise ...Each of these regions was affected at a different time by COVID-19, so we defined the COVID-19 event windows...Pytorch, which gave us the maximum likelihood estimates of each parameter. We approximated the data likelihood...

...North America. I hope that these perspectives, the optimism they encapsulate, and the solutions they promote...promote help motivate you to spend a little time advocating for science and getting yourself out there for...has been here before.  These attacks on science ultimately galvanized scientists to rally in Ottawa to protest...Institute. He also studies energy markets in his spare time. You can follow him on twitter at @thompn4. New ... Saturday in the tourist-trap nightmare that is Times Square. But I made the sacrifice in April because... entire scientific process.  I've spent a lot of time debating how to fix this.  As scientists, we work...lab where the groundbreaking research happens, sometimes forgetting how to engage with friends, family,...

... studies enabled by fluorescent proteins The optimization of GFP and its derivatives by the late Roger...Roger Tsien changed everything. For the first time, fluorescent proteins gave researchers the ability to ...visualize how cells interact after a particular stimulus. Using an epifluorescence microscope and GFP fusion...example, by harvesting lymph nodes at different times after antigen challenge, scientists were able to...to give a clear view of cellular movement in real time. Two-photon microscopy was the next revolution in...the ability to monitor cellular movements in real time. This technique was first Introduced to immunology...migration and movement by marking cells at a specific time. Photoactivable fluorescent proteins, for example...