...growth rate, so my cell pool is going to evolve over time, which is bad news for repeatability and bad news...had never done it before and it worked the first time probably because I used a robust cell line. If your...The Surveyor kit had a lot of good tips for first time users, all of which I followed. If you have never...the duplex and run it as an undigested control. Sometimes the PCR products can look smeared, so having an...products (GFP-GFP duplex) as a control. 4. After optimizing the reaction and confirming the presence of the...landscape of research opportunities, it’s an exciting time to be in the lab. Based on the power of available...John Doench & David Root and are described in Optimized sgRNA design to maximize activity and minimize...

...Did you catch our April AAV webinar with Tim Miles, PhD, Director of the CLOVER Center at CalTech? If...webinar! Special thanks to our panel of experts, Tim Miles, David Goertson, Xinhong Chen, and Miggy Chuapoco...didn’t get answered live - but he kindly took some time to address all your unanswered questions via text...Chuapoco, and to all our participants for their time and excellent questions.    Additional Resources...Adenoviral delivery of CRISPR/Cas9 Tips for a first time AAV user Resources on Addgene.org Find Adenovirus...

...Robert J., Mimee, Mark, and Timothy K. Lu. “Sequence-specific antimicrobials using efficiently-delivered...remain in the delivery of these agents, CRISPR antimicrobials could become our newest line of defense against...the growing problem. Above all, we need new antimicrobials, ideally ones for which the development of ...process essential to most bacteria, custom CRISPR antimicrobials can target specific sequences in a single virulent... Bikard et al. developed sequence-specific, antimicrobial, plasmid-based, CRISPR systems (Find the Citorik...particles using a helper system. These CRISPR antimicrobials successfully resensitized a beta-lactam resistant...observed substantial resistance to the CRISPR antimicrobials, and they suggest that targeting multiple resistance...

...Therefore, codon optimization may disrupt the fine-tuned timing of translation and ultimately protein function...Synthesis NovoPro Labs Codon Optimization Tool (ExoOptimizer) Genewiz Codon Optimization  ATUM DNA2.0  IDT Synthetic...Nakamura et al., 2000).  What is codon optimization? Codon optimization is an approach in gene engineering...when do you want to codon optimize your gene of interest? Codon optimization has become extremely useful... worth codon optimizing a human gene for expression in E. coli. Genes are also optimized for expression...codon optimized membrane protein expressed in mammalian cells. In this study, codon optimization resulted...and concerns: When not to codon optimize? Although codon optimization increases protein production in ...

...blocking time! Fill the time with smaller tasks, or simply enjoy some well-deserved down time while waiting...Temperature and time You may need to optimize your antibody incubation temperature and time. Though the most...talk about western blot optimization and troubleshooting.   Before you can optimize or troubleshoot a western...long transfer time. Be aware that the transfer works by electricity, so long transfer times increase the...blocking times or higher concentrations of blocking buffer. If you see a low signal, you can optimize blocking...backgrounds.  Once you’ve optimized antibody concentration, you can optimize ‌incubation conditions to...important to let them wash for the full amount of time, every time. Imaging The last step in a western blot tends...

...was repeated two or more times in tandem. Such plasmids are known as multimers, and they are the result... in a process known as plasmid multimerization. Plasmid Multimerization Early studies on plasmid topology...various multimeric forms (e.g. concatemers, dimers, trimers, tetramers). Since then, multimerization has ...plasmid multimers function just as well as monomers. However, due to their larger size, multimers may exhibit... detecting plasmid multimers. Digests themselves cannot be used to detect multimers, since restriction...frequency of multimerization, and in some cases completely prevent the formation of plasmid multimers (Bedbrook...a lower temperature such as 30°C or optimizing the incubation time can help maintain cultures in log phase...

...than not!), we think it’s about time we updated this post. How much time does it take to make a plasmid...traditional “one at a time” approach. According to his math, it takes five hours of hands-on time to make a single...decrease plasmid production time and cost If you’re short on money or time, there are a few options you...order to preserve time and money for their own experiments. Keep good records Oftentimes, you’ll be able...how long it takes to make a plasmid? Or how much time you have to spend cloning before you can start your...What about all the reagents you need to order? Sometimes, it feels like an eternity of cloning, waiting...shared milestone in 2018, we wrote a post on the time and cost required to make a plasmid. In 2023 (and...

...just end up spending more time in the lab. Obviously I didn’t have the best time management skills, but ... manage my time. As a busy researcher, you’ve probably already started coming up with time management ...planning, and procrastination. Time management through prioritiziation Every time you begin a task, you’re ...specific amounts of time to specific tasks beforehand and assess whether that time was sufficient for ...then you should definitely allot more time for that task the next time around. Finally, when prioritizing...some help. Today in our transferable skills guide: Time Mangement for Scientists. Back when I used to do...experiments in graduate school, I had to divide my time between lab work and practicing my SciComm skills...

...your plasmid yield is sub-optimal? If you are consistently getting sub-optimal plasmid yields from your...it will likely save time in the long run! While the choice of strain will ultimately depend on the particular...incubation time can improve the cell density and therefore the plasmid yield. You can optimize your growth...more to achieve maximum plasmid yield. The optimal growth time should be determined for each plasmid/strain... post was updated on March 21, 2018. Most of the time, plasmid prepping is a breeze. You get your stab...your prep, try our tips below! How to optimize your plasmid prep We'll discuss the following techniques ...strain to see if additional steps are needed to optimize yield. You might want to include an extra wash...

... Working with plants doesn’t always have to be a time-consuming process. While developing transgenic hairy...transformation process could save the plant biologist some time… months, in fact. In the Lee-Parsons lab, we are...plant, the Catharanthus roseus (Figure 1). The ultimate goal of our research is to enhance their production...C. roseus by working with young seedlings and optimizing several other aspects of the transient transformation...available for your species This will save you a lot of time. Don't re-invent the wheel. Test different leaf ...

...localization changes over time.Hallmarks of fluorescent protein timers When FP timers are first expressed,...expression time. 2. Timer functionality with a single gene Although it is possible to make a timer system ...FPs, the FP timers, add an entire new dimension to monitoring localization; using FP timers, researchers...population of timer indicates the age of the pool: the higher the ratio, the older the pool. FP timers have been... hallmarks of ideal FP timers: 1. Ratiometric determination of expression time The ratio of mature to ...immature fluorescence from the FP timer is dependent on the total expression time but independent of protein ...total expression time can therefore be used to determine how long a pool of FP timers has been expressed...

...hopefully help guide policies and ultimately treatments in a timely fashion.”  With this in mind, Bourque...supporting their access to useful resources during this time. Facilitate collaborations within the scientific...highly skilled scientists, willing to volunteer their time and expertise for this cause: from virologists who...

... slowly after introducing a cut, with residence times on DNA of approximately 6 hours. To our surprise...homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery." Elife 3 (2015): e04766...

...determine when the optimal time to conduct an experiment begins. This can save you time in the long run ...information.  Optimize titer for the DIO/FLEx vector Once you have determined the optimal injection parameters...begin to optimize the DIO/FLEx vector. This step is essentially the same as what was used to optimize the ...used.   Following this stepwise optimization will take a fair amount of time, and it may be tempting to skip...experiments. However, by optimizing all conditions first, you will save time, frustration, and funds in...the causes of off-target expression and ways to optimize experiments to minimize it. Overview of DIO/FLEx...created with BioRender.com   Strategies for optimizing expression So now that we know spontaneous recombination...

...level of intimacy that creates the magic of a safe space. No matter why, now is a great time to find a...work-from-home and just now realizing that we can’t bide our time and wait for it to be over to work on career skills...speaking For almost any science career, there will be a time when you need to speak publicly to communicate your... by now that people get jobs at least 85% of the time because of relationships. It’s much harder to build...

...which takes time because the solution is viscous, or by autoclaving, which also takes time. Rather than... virus for my first experiment. It was a bit intimidating, but I had my lab’s protocols and some great...total, this process takes 4-7 days, not counting the time needed to expand the HEK293 cells. Check out the...screening for ITR deletion is still necessary. To save time and money, I like to miniprep 5 mL of my AAV transfer... my AAV packaging PEI transfections. It saves me time and helps me figure out if I have enough plasmid...the HEK293 cell culture supernatant because it’s time consuming to prepare the 40% PEG solution. PEG takes...AAV lysis buffer and freeze/thaw the lystate four times in a 95% ethanol and dry ice bath to release the...

...selected sgRNAs in this optimal window and ranked them based on their optimized sgRNA rules and potential...genetic screen. However, there are benefits to optimized libraries that utilize only a few sgRNAs per gene...Sanson et al., 2018). These libraries utilize optimized sgRNAs that were designed based on the rules identified...library performance. The Root and Doench lab’s optimized genome-wide CRISPRko, CRISPRi, and CRISPRa libraries... a look at the features and advantages of the optimized human CRISPR libraries. Brunello: human CRISPRko... Thus, the Brunello library proves that a few optimized sgRNAs is enough for efficient library performance...Overall the Root and Doench lab’s have proven their optimized, human genome wide CRISPRko, CRISPRi, and CRISPRa...

...this adds up to a significant time savings. You can also save yourself time if you're transforming a plasmid... each week at Addgene, so we need to be cost and time efficient. We do this by making our own competent...antibiotics to give the antibiotic resistance gene time to be expressed.  However, you can skip this incubation...

...accumulated over time? Undergraduates can help to answer this question! The Pathways Over Time project The...The Pathways over Time project provides a framework for these undergraduate research questions. The idea...from a common ancestor ~350 million years ago. Over time, the two genomes have become very different due ...species diverged. Tips on using the Pathways Over Time plasmids The plasmids are used as part of a semester-long...D.M., Ludlow, L.H., & O'Connor, C.M. Pathways over time: Functional genomics research in an introductory... pathways over time...

...only easily identify base editing events in real-time but also enrich for base-edited cells in their experiments...Monitoring APOBEC and Cas9-mediated editing in real time The Harris lab created ACE, a reporter that monitors...deaminase proteins) and Cas9-mediated editing in real time (St. Martin et al., 2018). The ACE reporter is a...canonical base excision repair enzymes. At the same time Cas9n cleaves the opposing DNA strain which results...reporter quantifies APOBEC-Cas9 efficiency in real-time, it relies on DSBs which is not ideal for monitoring...efficiency of APOBEC-Cas9 editisome complexes in real-time (St. Martin et al., 2019). To create the eGFP reporters...sequencing (Standage-Beier et al. 2019). TREE is a real-time, fluorescent based identification system for the...