...value I could bring to potential employers. Over time, I discovered that I not only enjoyed the technical...a cell culture experiment myself would take less time than the seemingly burdensome task of explaining...strengths, and learning styles. For example, during my time as a lab scientist, I worked with a diverse team...when managers fail to set expectations or provide timely feedback, leading to unproductive dynamics. I experienced... experienced this firsthand when it took me some time to adapt to the communication style of a new, junior...solid foundation in the basics of management. Invest time in understanding key principles through books, workshops...

..., these methods can be labor intensive and quite time consuming.   As Cas9/gRNA activity is crucial for...need in the scientific community for efficient real-time Cas9 activity assays. Recently scientists have created..., integrated reporter for assaying CRISPR/Cas-stimulated HDR activity) The Madison lab created SRIRACCHA...ratio of RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  By using the RFP:GFP...reporter systems provide efficient, rapid, and real-time read-outs of Cas9/gRNA efficiency. So if you are...integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair. Journal of Biological...

...rigorous studies will give false signals from time to time. False positives in original studies and false... provided in most published papers. The precise timing, order, or every combination of materials and treatments...preregistration Preregistration is the process of creating a time-stamped research plan in a public repository before...control, and you think there are four important times to measure that growth rate, you report the results...occurs during the course of the study, say a fifth time has unpredictably high rates, should be reported...

...out of their optimally suited temperature ranges and make them grow better at suboptimal ones, which could...are your regular next-door microbes which grow optimally in moderate temperatures (usually defined between...quantity can significantly reduce the operational time for a given process. These are extremely useful ...worth the trouble.  Enter ELSA: E. coli Learning Suboptimal Acclimatisation To resolve this impasse, our ...mesophilic chaperones would not function well under suboptimal conditions. This means recombinant proteins can...

...tools. How many times have these plasmids been shared in total? Just over 49,000 times! Their long-lasting...adventures...From moving office locations three times, to opening our UK office in 2014, to starting our...such as our second most requested plasmid of all time, pMD2.G. Find psPAX2. pMD2.G - Also from the Didier... use in human cell lines, the Zhang lab codon-optimized SpCas9. Using the BbsI restriction site, a pair... version is the 15th most popular plasmid of all time, but was found to have a point mutation in the puromycin...

...transcription >1,000-fold when activated. Lag time The time needed to induce a given promoter varies. Promoters...a repressor/inducer will have higher lag time due to the time needed for transcription/translation. Many...transcription of a target gene, it therefore determines the timing of gene expression and largely defines the amount...complexed with cAMP activator protein (CAP) can also stimulate AraC binding to I1 and I2 sites. Supplementing...

...as many journals as possible at the same time. Take your time picking the appropriate journal and explain...subgroup of researchers in your area of expertise? Sometimes, the funders of your research may stipulate that... both the referees (as they get credit for their time and effort) and the scientific community, as the... you are sending it to. It’s wasting everybody’s time if you don’t. Add a clear cover letter to your submission...published. In summary, it is a good idea to take some time and look into which journal would be best suited...

...write about your plasmids in a future blog post. No time to read? Listen to our hot plasmids segment on the...CyOFP1, a large stokes shift, long fluorescence lifetime, and a stable monomeric form as determined by ...resonance energy transfer) and FLIM (Fluorescence Lifetime Imaging Microscopy) applications. To demonstrate...isolated and characterized: mScarlet (fluorescent lifetime of 3.9 ns, quantum yield of 0.70), mScarlet-I ...maturation in cells compared to mScarlet, fluorescent lifetime of 3.1 ns, quantum yield of 0.54) and mScarlet-H...photostability compared to mScarlet, fluorescent lifetime of 1.3 ns, quantum yield of 0.20). All three mScarlet...working with edited “pools” of cells, eliminating the time-consuming step of single-cell isolation. Agudelo...

...the myriad challenges we encounter. As you spend time in a lab learning techniques and taking on independent...assessed potential protocol modifications, and ultimately decided on the changes to implement. As with ..., there will always be room to improve. The next time you face a problem, try to be mindful of how you...yourself to think of more creative solutions. Ultimately, problem solving is less about technical know-how...both technical and transferable (i.e. teamwork and time management), your ability to solve problems efficiently...Collaborating with others outside of your organization Time management Managing a team Leadership Resources...

... their cargo can begin, which all takes time. Though the time delay until expression is highly dependent..., Marlon R., et al. "Development and optimization of a real-time quantitative PCR-based method for the...AAVs tend to be very neuro-permissive (Issa, 2023). Time to expression AAVs have to infect cells, hijack ...target tissue, two weeks is the standard ballpark estimation. Once expressed, viral cargo is usually quite...

...tried-and-true tools means less time getting up and running and more time working on interesting biotechnology... a request for a particular plasmid and now it’s time for our lab technicians to retrieve the sample and...procedures. With this data in hand, we can go back and optimize our procedure to make the whole process work more...Barcoded materials make everyone's life easier, the time and capital cost of getting up to speed with a setup...Bioinformatic Analyses Use Software Apps to Save Time and Track Your Experiments Additional Resources...

...reading through an article for the first time. The first time through, just get an idea of the techniques...with the scientific literature a little bit at a time. Searching for literature My go-to search engines... the jargon and background in your field, but sometimes papers are also just poorly written. By nature...connection or flow among the figures and results that ultimately builds up to an overall conclusion(s). In some...literature in your field of interest. Set aside some time each day or week to at least scan the titles of ...

... are financially accessible. Sometimes only a few hours of coaching time can make an enormous difference...volunteer mentors are only able to go so far with their time and advice. A professional coach is paid to carry... can take some research.  It helps to spend some time thinking about what your goals are for the coaching...Coaching. One nice thing about a group is that sometimes this offers the option of matching with the best...a good match as a coach for many scientists.  Sometimes it can be helpful having a coach that can offer...

...have no idea how to write a grant!”, “I don’t have time for this if he wants data by next week!”, and “This...administrative side of the grant to be established. Take time to read and fully understand the RFA. The RFA contains...current state of the art technologies. Therefore, take time to review the literature and offer educated insight... will address the research challenge. Now is the time to make bold statements. While some of the statements...supplies, personnel, equipment, travel, etc. As a first time budget writer, you may find this section challenging...feedback on the application. Make sure to invest time and consider the reviewer's comments. Whether the...

...write about your plasmids in a future blog post. No time to read? Listen to our hot plasmids segment on the...arrestin recruitment assay incorporating 314 codon-optimized GPCR sequences for human cell line expression....directly for drug screening or easily shuttle the optimized, expression validated GPCR sequences to any desired...strains remains seriously limited, as prior optimizations are required for each individual target species...being generated, there is a need for genetic optimization of industrial yeast strains. However, industrial... the well-known yeast two-hybrid assay are not optimal for studying native mammalian protein interactions...transfer vector for in-vivo expression and plasmids optimized for dCas9-VPR expression in drosophila and yeast...

...: you only need to express two gRNAs at the same time. One system you could use is pX333 from the Ventura...follow these same assembly principles, but they’re optimized for different purposes.   Gersbach Lab multiplexing...amplified for cloning into one of three Zeamays codon-optimized Cas9-containing binary vectors. The system is ...destination vector based on pCAMBIA. Cas9 is plant-optimized, and plasmids are available for both monocot and...incredibly fast protocol, with only 5 hours incubation time needed for a single round of editing, and a subsequent... paper at Addgene. 10. Port, Fillip, et al. “Optimized CRISPR/Cas tools for efficient germline and somatic...Ronda, Carlotta, et al. (2016). "CRMAGE: CRISPR Optimized MAGE Recombineering." Sci Rep. 6:19452. PubMed...

...standardizing the process. When we started, too much time for this project was spent selecting guides, designing... CrispyCrunch While CrispyCrunch may evolve over time, you can depend on it to stick to a few guiding ...Furthermore, we did not find any tools which were optimized for HDR, except for TagIn. HDR requires the additional...enabled it to return guides and primers in batch, optimized for HDR. Compared to wealth of resources for guide...insert, the more poorly the guide performs HDR. Optimal mutation in-depth One the most sophisticated features...mind, we implemented the following algorithm for optimal mutation in CrispyCrunch: If the guide PAM resides...

...allowing you to spend less time researching plasmid basic features and spend more time developing the clever...as a means of detection; they are useful almost anytime you want to express multiple genes in the same ...the genes expressed from the same cassette is sometimes advantageous, particularly when only a portion... be feasible to express more than two genes at a time using IRES elements. Further, scientists have reported...Likely the choice of specific 2A peptide will ultimately depend on a number of factors such as cell type...

... a 2D barcoded tube (see next section) and every time something is removed from this tube, the barcode...up samples as we transform dozens of samples at a time.   Not everything that is received is immediately... Most of our samples (98.2%) transform the first time and can be stored right away. Every week, the lab...better for tubes that are meant to be read one at a time. 1D barcodes can be purchased from many companies...prevents us Addgenies from wasting hours of valuable time searching for samples and keeps us from discarding... samples get lost during their journey. Take the time to track your samples today and never lose a sample...

...and translating a protein fluorophore also takes time - bypassing translation altogether with an oligonucleotide-only...oligonucleotide-only sensor could save precious minutes in a time sensitive detection assay. In 1999, Grate and Wilson... a non-protein based visualization technique. Ultimately, MG proved to be too toxic for many in vivo applications...aptamer, called Spinach, required longer exposure time than its protein equivalent and was less stable ...throughput assays with microarrays and biochips. Ultimately, there are just some applications that lend themselves...imaging of RNA processing using broccoli reveals optimal aptamer expression strategies.” Chem Biol. 2015...

... lot of great output in a short amount of time can sometimes become cauldrons on the verge of boiling....inform a go/no-go decision in a very short amount of time. Such a decision determines whether the client invests...achievable tasks, and then executing on them in a timely fashion while allowing for creative solutions to...services that help diabetic patients manage doses and timings of their drug treatments. The process of rising...goal posts” as a part of a case, consulting can sometimes be challenging. The fast paced nature of projects...

...repository through our Hot Plasmids articles. Today, it's time for another edition of Hot Plasmids!  Here's what... size-limited AAV delivery. PE6c and PE6d are optimized for dual-AAV systems. These variants use different... tagged iPAK4 from different promoters, specific time points appear as fluorescent bands in the fiber,...tape” recording. The authors created externally-timed bands of color with HaloTag-iPAK4 by applying different... accurate to within 40 minutes. Figure 4: Time-resolved protein ticker tape. A) Design of iPAK4...iPAK4 plasmids here! Lin D., Li X., et al. (2023). Time-tagged ticker tapes for intracellular recordings...

...with proper protein folding, and deal with sub-optimal codon usage (Table 1) A few mutations are common...reductase to faciliate proper folding; may increase multimer formation Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuII ...as IPTG is added to the growth medium. Induction timing is important, as you typically want to make sure...reaching the desired density within a reasonable time-frame. For these cases, some strains carry an additional...experiment to test variables such as temperature, time, and media conditions. Many recombinant proteins...many proteins, a rich media such as TB or 2XYT is optimal because of the high cell-density they support; ...

...photocurrent vs. time. The resulting histograms is described through maximum current (H), the time the cell needed...of a histogram, which graphically represents the time and intensity of the cell’s illumination (see Figure...maximum signal strength, while W results from the time that the cell spent passing through the laser beam...clearly distinct positive and negative populations. Oftentimes, you’ll be presented with a trend of two populations... With our first three gates out of the way, it’s time to move on to my experimentally-specific gates. ...

...pool down to plasmids expressing variants with optimal function. High-throughput DNA sequencing is then...concentration of Taq polymerase, 2) increased PCR extension time, 3) increased concentration of MgCl2, 4) increased...Wrenbeck et al for details on how nicking was optimized. How should I design my mutagenic primers? The...orientation of the BbcVI restrict site matter? Most of the time no, but there are 2 instances where it does. 1) ...saturation mutagenesis method are 1) ease of use, 2) time savings, and 3) ability to perform single site or...Stapleton, Adebola Adeniran, Keith E J Tyo, and Timothy A. Whitehead. "Plasmid-based one-pot saturation...

... Taking control of a RhoGEF is a great way to stimulate and study these physical processes. Joachim Goedhart...inhibit it. Restricting the effect to a specific time or location can be critical when, say, the gene ... protein’s function at a very specific place and time. Future studies with these CIB1-Rabs in other contexts...variants called SuperPLDs with 10, 30, 50, and 100 times the activity of the original, generated using directed...monitored by phosphatidic acid probe GFP-PASS. B) Time series of HEK 293T cells expressing plasma membrane-targeted.... D., & Goedhart, J. (2023). Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho ...

...share my experiences from my own job search and my time on faculty search committees. Having successfully...you think you can accomplish. No matter how many times I say this, candidates regularly try to convince...The second question is whether there is chalk. Sometimes. It can also be a slide deck, markers, etc. Chalk... them into simple goals/outcomes. This isn't the time to go into detail. Finally, state how successfully...the aims of a chalk talk. Questions take a lot of time to discuss and the questions are important. However...secretory proteins in the Endoplasmic Reticulum, optimization of fluorescent proteins,  and live cell imaging...

... to alleviate those differences, they had a hard time maintaining their friendships. I put “neutral” in...ability to remember the right stories at the right time. Like many of the posts in our Transferable Skills...address these types of harmful conflict early, because time frequently does not heal these types of wounds. ... know about those particular teams at that given time. I think about how previous iterations of those ...collaborating, and compromising. Though there may be a time and place for avoiding, competing, or accommodating...looking for a promotion, have some solid examples of times when you have used your empathy, listening skills...

...incredibly fast protocol, with only 5 hours incubation time needed for a single round of editing, and a subsequent...other advantages including shorter plasmid curing time. pCRISPomyces for editing in Streptomyces  Streptomyces...identification of a new CRISPRi tool, they also optimized the system by modulating the PAM to further tune...-Cas nucleases to produce sequence-specific antimicrobials. Nat Biotechnol 32:1146–1150 . https://doi....RJ, Mimee M, Lu TK (2014) Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases... Sommer MOA, Nielsen AT (2016) CRMAGE: CRISPR Optimized MAGE Recombineering. Sci Rep 6: . https://doi....difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials. mBio 11: . https://doi.org/10.1128/mbio.00019...

...culturist has to deal with this problem. It is estimated that mycoplasma is responsible for up to 60% of...air if the cell line remains exposed for a longer time. The aerosols are invisible to naked eyes and hence...Mycoplasmas can remain in the dry state for a longer time. When they come in contact with a nutrient source...continuous culture for at least two weeks to give time for low levels of contaminations to grow. For testing...products or reagents that have been stored for a longer time. Use fresh media as much as possible and the ones...ways to rescue the cultures. Elimination is a very time-consuming process and increases the risk of secondary...