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...Hurt. Understanding how individual parts can be assembled into TUs is key to understanding MoClo. By...another optimized platform. Gene Stacking As the name implies, gene stacking is the use of a combination of...transcriptional units, and multigene constructs. Let’s start by examining individual parts and transcriptional...plant biology to achieve traits such as disease resistance. MoClo is a great tool to deliver many combinations...MoClo Interested in using MoClo? You don’t have to start from scratch! As you can imagine, there is an array... many different MoClo kits have already been established. Keep an eye out for our next blog on choosing... Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS One. ...

...to minimize rearrangements such as NEB Stable or use a standard strain, like DH5α, grown at 30 °C instead...save you time, and allow you to standardize your experiments later. Stay tuned to the Addgene blog and ...that have helped me troubleshoot some technical obstacles when working with virus: 1. Viral vectors undergo... to a target cell by reducing the repulsive electrostatic forces between these two negatively-charged ... will be selectable with the antibiotic whose resistance gene is encoded in the viral vector. This antibiotic... may be some low-level fluorescent protein or resistance marker expression following transfection, higher-level...Central PMCID: PMC84922. 5. Ugai, Hideyo, et al. "Stability of a recombinant adenoviral vector: optimization...

...and distance that prevents interaction with each other and the audience. Put the panel on a stage if the...Check out Joanne's Reddit AMA Perhaps we should start with some examples of a bad panel discussion? You...relaxed phone call than when first confronted on a stage in front of a crowd. The moderator will then develop...You can use this to the advantage of the program. Start the program by polling the audience with a few questions...with some written poll questions to get things started. Give a couple of audience members a chance to ...science organizations, I asked audience members to stand and describe unique nonprofit models that differed...possible, make sure the panelists are prepared to stay around to meet and greet audience members. Having...

... be needed to achieve your intended next career stage and create a timeline for achieving these goals....A project with sufficient preliminary data to establish your own lab and get an independent investigator... grant (e.g. an R01 from the NIH, in the United States) Strong letters of reference that attest to your... annually advertise for Group Leader positions starting in August, the year before the position would ...you're using a new protocol or reagents, do you understand all of the steps? Do you know how the reagents...taking this time, you can help ensure that you understand the methodology and can have confidence in the...burnout The suggestions in this blog add up to a substantial amount of time- most of your waking hours. This...

...Kostman.   Important considerations before you start an experiment with CRISPR The hammer, the jigsaw... an alternative ATG downstream of the annotated start codon. Likewise, we avoid target sites that code...undertaken lightly! Indeed, truly achieving the gold standard requires not one but two rounds of single cell...in a 5 - 10 nt window relative to the PAM, and bystander edits are possible if there is another target ...target a ~100nt window upstream of the transcription start site (TSS), while for CRISPRi, a ~100nt window downstream...the target (e.g. accessibility due to chromatin status). No computational prediction is ever perfect, ...nevertheless still lead to activity has been established, and can be used to identify sites that are likely...

...for speeding things up and for standardizing the process. When we started, too much time for this project...for improving it. As much as possible, it uses standard tools and databases, including primer3, biopython...scientists of the burden of having to manage all the standard information that goes into a 96-well plate and...by specificity of targeting or by cut-to-insert distance for HDR. (The notable exception is TagIn––see ...CFD score) * (gaussian normalized cut-to-insert distance) This gives each guide a score ranging between...used in the rank equation above. Cut-to-insert distance was fitted to experimental data of its effect ...rank equation above. Generally, the greater this distance between the cut and the insert, the more poorly...

...experiments. Fluorescence is the emission of light by a substance that has absorbed light. The emitted light is ...wavelength can change. In a few cases, the initial state of the protein is non-fluorescent, thus allowing...another FP (e.g. YFP). FRET only occurs when the distance between the two FPs is <10nm, and should be considered...intensity can be affected by the temperature. For instance, enhanced GFP (EGFP) was optimized for 37°C, and...0.1) and this should be taken into account. Photostability can be affected by experimental parameters ...excitation light intensity, pH or temperature). Photostability: Fluorescent molecules gets bleached (i.e. ...after prolonged exposure to excitation light. Photostability can be as short as 100ms (EBFP) or as long ...

...bacterial resistance marker used to grow plasmid containing bacterial cultures Bacterial Resistance: Use this...producing and purifying it in your lab. Instead of starting a new search or clicking through to a different...Materials or elsewhere in their papers. Maybe they started their deposit prior to publication and were able...searching with filters Not every plasmid search will start from a publication. You may have an idea for a plasmid...express ubiquitin under control of a CMV promoter. Starting with two search terms we see there are plenty ...need to apply different filters you do not need to start a new search. Simply click the ‘X’ next to any filter...terms with gene and guide RNA / shRNA inserts Resistance Marker: This indicates the selectable marker ...

... D., Stamnes, S., Holt, K., Sinn, P., May, A. P., & Paulk, N. K. (2020). Methods Matter: Standard Production...neuroscience Let’s consider my first challenge when I started working with AAV vectors. I wanted to use an AAV...spread. (D-F)  High-magnification images immuno-stained against dopamine beta-hydroxylase (DBH, magenta...Addgene Data Hub is designed to help scientists understand how our vectors perform in various experimental... end of the year, those that meet our minimum standards (clear and detailed methods and image descriptions...available through the Data Hub. These minimum data standards are designed specifically to allow scientists ...Gao, G., Mueller, C., & Flotte, T. R. (2015). Stability and compatibility of recombinant adeno-associated...

...COVID-19 cases each week. Statistical Modeling Note -- If statistical modeling isn’t up your alley,... estimated total of 380,000 deaths worldwide (statistics provided by Google on 6/4/20). Many countries..., feel free to skip directly to the results. To start modeling our data, we first chose to re-examine ...order several plasmids at once, breaking the statistical independence assumption of the Poisson process...dataset) for the U.S. and Europe. To improve the stability of our model, we considered only the 10 papers... least impacted. These results highlight the instability and uncertainty in estimating parameters from...investigation of model fit revealed some potential instabilities in the China data, so we recommend interpreting...

..., each flanked by a transcription start signal sequence (gene start) and a transcription termination/polyadenylation...used to develop an understanding of and manipulate the biology of EBOV. For instance, the roles of specific...luciferase or eGFP) flanked by the EBOV-specific gene start and gene end sequences. Thus, the minigenome contains... of Minigenome System). From a more technical standpoint, a plasmid with all four genes and necessary ...luciferase). The polymerase recognizes the gene start signal and initiates transcription of the reporter... the T7 RNA polymerase gene or using cells that stably express T7 (e.g. BSR T7/5 cells) (1). We hope that... that minigenomes will greatly enhance our understanding of both basic virology and the functions that...

...there are more! Engineering disease resistance through gene stacking, increasing production of a protein... uses of MoClo and the kits Addgene offers to kickstart these applications, no matter what your model ...isolation, then the MoClo Pichia Kit will get you started (Obst et al., 2017)! It is specifically curated...reporters (Engler et al., 2014). This kit is a great starter for your general plant cloning experiments. Addgene...basics’ MoClo plant kit buddy). It is important to understand your experimental needs before choosing one or...with a large library of common parts to get you started quickly (Fonseca et al., 2019). Recently, the toolkit...that backbone differences such as the order, or distance in the placement of plasmid elements can impact...

...inputs, such as the file name. We started small with a print statement and tested that the desired data...associated publications, but our quality control (QC) standards have evolved over the years to keep up with the...     Fig. 1: QC'ing data at Addgene is like standing under a Niagara Falls sized rainbow   Over...problems was deciding on the right system for us to start using. Prior experimenting with open source assembly...perform:      Fig. 2: Quick-stepping to a FASTA file   With our outline of individual steps ...Creating a bioinformatics workflow in Nextflow Establishing a complete pipeline however, required more than...libraries such as Pandas, Numpy, and Biopython.  We started at the usual recommended place: documentation. ...

...regulatory elements or use a non-canonical code (Gustafsson et al., 2004), such as Hoogsten base pairs and...could affect protein conformation, folding and stability, change post-translational modification sites ...actually be necessary for proper protein folding (Stadler and Fire, 2011). Therefore, codon optimization ...translation initiation (Dresios et al., 2006) and mRNA stability (Hausser et al., 2013) to name a few. This could... you may want to codon optimize before you get started. There are a ton of online resources available ...Biol 13:30–34 . https://doi.org/10.1038/nsmb1031 Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias...tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Research 28:292...

...electrophoresis (PAGE) followed by silver staining or SYPRO Ruby staining and the molecular weight and relative...after negative staining. Empty vector particles can be identified after negative staining and appear darker...lentiviral preps.   Viruses are generated using standard methods that have been optimized for each specific...three fold higher than those achieved using the standard qPCR method.  Titering by the University of Pennsylvania...high quality viral vectors by optimizing and standardizing production protocols and performing rigorous...of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV subjected to ...production are propagated in the endA-mutated NEB Stable strain of E. coli. In addition, plasmids are typically...

.... I started watching the questions and preparing answers early in the day. Although I didn’t start answering...people or discussing science, it is a great way to establish yourself as a thought leader and have real conversations...Software for Biology) on Reddit and got some substantial traffic that we thought it would be a good idea...definitely glad we did - you can see some of the stats from the AMA to the right - the Reddit community...description of our AMA and Joanne’s preferred time to start answering questions. r/Science has pretty strict...was posted at 8:00 am and Joanne didn’t have to start answering until 5 pm). The ability to see the questions...Fortunately, Reddit Science is heavily moderated. I started watching the questions and comments go up early...

...experiments and understand their data. Likewise, we want to contribute to helping others understand ours. Improving.... The PLOS policy on data access is a formal statement that is already practiced or required by many ... errors in assumptions, use of inappropriate statistical methods, omission of some data, etc.  The firestorm...published, this data is usually condensed into a statistically evaluated set of values.  However, to evaluate...sharable form is indeed a significant effort. Our standards of data recording are highly variable and usually... images are said to be representative or the statistical variation is calculated and placed in the legend...are used to publications presenting a polished, standard and usually limited view of data. In addition ...

...with physical map distances of 75 and 2 Mbp, with the calculated fluorescent distances correlating with...organization of the nucleus in space and time, and to understanding how nuclear organization varies across the life..., or how organization may be altered in disease states. dCas9 gets a colorful makeover Even without its...with the previously established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that...degree of chromatin compaction even for this small distance! To the authors’ knowledge, this work represents...applications Ma et al. developed this technique using standard fluorescence microscopy, and are thus limited ...priority, as researchers work to increase our understanding of chromatin organization. This fluorescent ...

...various stages in their careers to get advice on choosing a lab, picking your research topic, starting a lab... hard work, collaboration, and staying honest with personal goals. Stay tuned for the follow up blogs ... lab, and more. Before we start, we'd like to extend a special thank you to Dr. Tom Ellis (Imperial College...with full force. George Church 1. “Initial and sustained motivation is a huge component of success.” 2... "Choosing Your Research Project," “Advice for Starting Your Lab,” “Fun in the Lab!" and more.   Acknowledgments...Choosing a Research Project Advice for Scientists Starting a Lab ...

...bluntly with WT Cas9 and one gRNA, you can create a staggered cut using a Cas9 nickase and two gRNAs.  For ...test nickase preferences using HEK293 cells. They started by comparing D10A and H840A total editing efficiency...PAM-in and PAM-out configurations with varied nick distances (40 - 130 nt). From these results, it’s clear ...than that of H840A, especially with smaller nick distances. Using D10A, they subsequently showed that editing...low when the gRNAs are too close (7-23 nt nick distance). Both D10A and H840A are potent editors, but ...obtain high HDR efficiency. Nickases create a staggered cut - could this system mediate repair throughout...the target site rather than the optimal <10 nt distance. Employing two gRNA sites that are further away...