...think “but that won’t happen to me, I have a backbone”. The sad truth is, if it happened before, it almost...choosing a lab, but almost all of these guidelines can apply to any workplace or job. It amazes me when the most...scheduled or only ad hoc? Does the mentor keep appointments with group members or are they frequently cancelled...conflicts in the lab? Do they motivate with the appropriate balance of encouragement and criticism? Do they...was told that the PI removed a woman from the appropriate first authorship position on a publication of...and profoundly regretted it when the same thing happened to her a year later. I hear too many science trainees...almost certainly will also happen to you. As you speak to lab members and alumni, don’t ever disregard warning...

...t just happen to you or me (though, let's be clear, they have all happened to me.) They happen to everyone...is, when it happens to other people, it’s normal and quickly forgotten, and when it happens to you, it’...communicator, I’ve done a lot of public speaking, and I'm happy to share what I've learned along the way. Own your...comes to public speaking, Murphy’s law definitely applies: what can go wrong, will go wrong. The trash removal...interest incorrectly on slides 8-16. Your notes disappear. The wrong version of your slide deck gets uploaded...completely normal to be anxious, nervous, and/or apprehensive. I still get nervous quite regularly, and I'...

...AID an appealing tool for studying essential proteins. Until recently, it was challenging to apply the AID-tagging...brief articles highlight the main features and applications of a partiular plasmid or set of plasmids. We...December 2016 (Q3) MARCH 2016Enhancer activity mapping with STARR-seq   Although many technologies have...classical TANGO assay, Kroeze et al. developed a new approach termed Parallel Receptor-ome Expression and Screening... (PRESTO-TANGO) to screen the NCC-1 library of approved drugs against the entire kit; a parallel analysis...multiple expression systems before they achieve the appropriate expression level for their gene of interest. ...GMAP-compatible vectors for a wide variety of biological applications. First, GMAP-compatible backbones for lentivirus...

... drug approval boards are being finalized, and it is very likely the treatments will be approved for use...disease. Meanwhile, Hans Clevers and colleagues applied CRISPR to disease correction in adult stem cells...CRISPR-based editing therapies that are currently approved or have made significant progress in clinical ...Therapies Cancer and CAR-T therapy The FDA has already approved CAR-T immunotherapy for cancer treatment by traditional...These therapies are delivered with an autologous approach, editing patient cells ex vivo and then returning...but especially concerning when using a systemic approach. Regarding effectiveness, if the editing frequency...meaningful way. Delivery, especially for systemic approaches, has its own unique set of obstacles. Thus far...

...which subpopulation of DA neurons could activate appetitive rather than aversive stimuli. In a recent paper...rAAV FLEx-based vectors to answer this question, mapping and characterizing the activity of the DA neurons...pursue a teaching career, and she now is a very happy scientific communication manager at genOway.     ... cell types for imaging and long-range circuit mapping." Journal of Neuroscience 28.28 (2008): 7025-7030...Philippe Coulon, and Ilka Diester. "Optogenetic approaches to study the mammalian brain." Current opinion...Yizhar, and Karl Deisseroth. "The development and application of optogenetics." Annual review of neuroscience...Mei, Yuan, and Feng Zhang. "Molecular tools and approaches for optogenetics." Biological psychiatry 71.12...

...Many thanks to our guest blogger Erik L. Snapp! Erik Lee Snapp received his PhD in Molecular Microbiology...This post was contributed by guest blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite...problem for fusion proteins. One of the major applications of FPs is to visualize the localization, dynamics... dimerize can form dynamic contacts that bring apposing membranes together and can wrap the membranes ...the Endoplasmic Reticulum and live cell imaging approaches. Erik is also an avid long distance runner, gardner... gardner, and cook.   References 1. Snapp, E.L., et al., Formation of stacked ER cisternae by low affinity...An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins. Protein...

... the appropriate CRISPR tools for your application: Wildtype Cas9 or Cas9 nickase are appropriate for ...well as suitable applications. Expression System Components of System Application Mammalian expression...CRISPR mammalian expression systems, the typical applications for each, and potential delivery methods.   ...mutations near the gRNA target site. Choose an appropriate expression system and delivery method: Do you...electrophoresis and/or next-generation sequencing more appropriate? If you already have an end product in mind...or more sgRNAs and Cas9 are required for any application. If you want to include a homology directed repair...

...Alternatively, you can run a reagent gradient. In this approach, you’ll run the same protein concentration in ...concentration of your reagent. Be aware that this approach requires excellent attention to detail, since ...down to voltage and time. Make sure to load an appropriate protein ladder on your gel and keep an eye on...too high. If you’re seeing a smiley face at an appropriate voltage (usually 10–15 V/cm of gel), your gel...box. If overheating remains an issue, which can happen with large proteins, try lowering the voltage and... buffer — so it’s better to start over if that happens.  Finally, if you’re seeing a weird issue and can...your reversible staining method of choice is appropriately sensitive. If using a PVDF membrane, you can...

...engineer various constructs with applications in flow cytometry approaches in mammalian cell culture systems...develop diverse fluorescent reporters is a useful application of high-throughput cloning. MXS Chaining is one...plasmids for fluorescence imaging or flow cytometry applications in mammalian cells. The modules used in this...transgene expression, or a variety of other clever applications. In one example, modular constructs generated...methods are suited for particular downstream applications. The tradeoffs between various cloning methods...from them. These enzymes might not, however, be appropriate for cloning genes from organisms with high CpG...quickly, and simply. We hope that you can similarly apply MXS chaining to your experimental needs. Let us ...

...amenable to transfection or viral transduction     Appropriate promoters must be chosen for both Cas9 and gRNA... Cas9-gRNA ribonucleoproteins? One alternative approach, which avoids many of these complications, is ...used for most of the current genome engineering applications of CRISPR including: generating single or multi-gene...pathways. This makes Cas9 RNPs useful for CRISPR applications where limited expression of Cas9 is required...vendor, this can often be expensive. An alternative approach is to express and purify His-tagged Cas9 from ...this technique in cell culture, it has also been applied to genome editing of mouse zygotes, through a process...inner ear (Zuris et al. 2015). A more targeted approach using RNPs has recently been described in vitro...

...structural proteins that make it possible, see the appendix DNA is a 3D fractal). Enhancer biology is a complex...acetylation) - tri_methylation In the next section we’ll apply the above formula to a real-world experiment. We...: the enhancers! This leads us to a three-step approach for designing an experiment to identify the critical...therapeutics. We’ll have more to say about clinical applications of CRISPR in the next section. In order to denote...mechanism for CRISPR. Therefore if a gRNA drops or disappears over time, we infer that the enhancer it targets...own CRISPR screens for enhancers, check out the Appendix. Conclusion: Shedding light on dark matter Precise...is the foundation of precision medicine. We can apply this knowledge to create targeted disease therapies...

...the candidate application process, the candidate’s ability to write a grant application through a written...funding opportunity coincided, and they stepped up to apply for a grant which lead to their new career path.... much more than writing. In fact, many grant applications and grant makers have moved away from long written...Now they require grant seekers to use online applications with significant character limits for the written...written portion of the grant application. They have increased their demand for stronger budgets with clear...duties can range from coordinating the grant application and submitting it, to evaluating the capacity...grant readiness, funding opportunity research, application writing and development, project implementation...

...activity of CcdB. Applications of ccdB in plasmid cloning Perhaps the most widely used application of CcdB is...used as biological tools for a wide range of applications in molecular biology or in vivo studies. Let...types of plasmid addiction systems and their applications. The discovery of toxin-antitoxin systems Toxin-antitoxin... by binding the GyrA subunit of DNA gyrase and trapping it in the cleaved complex. This results in DNA...construct. Other toxin-antitoxin systems and their applications MazE/MazF Aside from CcdB/CcdA, there are many...or diluted as the cell grows resulting in the appearance of unmethylated sites in the chromosome. The ...were retained in 75% of cells. They were able to apply this system as an orally administered diagnostic...

... QC/QA team as a DNA Sequencing Intern. The application process When I first saw the comprehensive job...for this co-op, I was immediately interested in applying. This had stood out to me because it gave me a...immediately clear that Addgene wanted to ensure applicants understood what the expectations were for the...the intended position. After submitting my application, I was contacted to participate in a brief fifteen...Friday, I received the official offer. I was so happy to hear back, I accepted that day and was excited...the depositing labs intended. With the use of applications like SnapGene and Geneious, I was able to determine...they would be disregarded - everyone was very approachable.     Fig. 1: Examples of DNA sequencing...

...substitutions (or deletions) in a tailored manner. The approach can be used in conventional cloning (to introduce...introduce or remove restriction sites), in mapping of regulatory elements (to mutate promoters/enhancers in...of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate genome editing... then, when the plasmid is digested with the appropriate restriction enzyme and run on a gel, one of the...ablates a restriction site, digestion with the appropriate enzyme will merge two smaller bands visible in...larger band (not shown). As a footnote; a similar approach can be used to identify Cas9/CRISPR-induced genome...template for the mutagenesis reaction. Alternative approaches to site directed mutagenesis Point-mutagenesis...

... AAV2 yields approximately 1.8-fold without affecting cell viability. This effect appears to be serotype...AAV yields. Given the wide range of downstream applications scientists rely on our AAV preparations for,...assays, in vitro transduction, and silver stain approaches we show that there is no difference in purity...so excited? For starters, we believe that this approach can be adapted by vector cores and laboratories...investigated 3 serotypes it’s possible that this approach will be beneficial for a host of other AAV serotypes...serotypes; it will be exciting to test this approach with different capsids. Most importantly, because .... "Substantial overproduction of antibodies by applying osmotic pressure and sodium butyrate." Biotechnology...

...unedited strand (Chen et al., 2021). The team applied this approach to both the PE2 and PE3 systems, naming ...and flexibility to the CRISPR editing world. The approach, originally published in Nature, is a “search-.... The initial result is a heteroduplex, with overlapping strands of edited and unedited DNA. The heteroduplex...editing typically needs to be optimized for each application, and the efficacy varies widely — it still almost...optimized enzyme can be used with any of the PE2-5 approaches, sometimes referred to as PE2max, PE3max, etc...improved efficiency, although both enzymes still approach or exceed PEmax editing efficiencies for short...prime editing. To protect the 3′ end, the Liu lab appended RNA pseudoknots, calling this longer RNA an “engineered...

... achieve multiplex IF. One of the most common approaches is to use sequential cycles of primary and secondary...detection with a mixture of primary antibodies and appropriate secondary antibodies. The general process is ... 1: Schematic of multiplex immunofluorescence approaches. A) Sequential Multiplex IF, where one target...multiplexed IF, many of the same considerations apply as for standard IF. However, there are a few specific...primary antibodies that have been validated in IF applications (find IHC validated or ICC validated antibodies... you have some complex patterns with several overlapping targets to interpret. Check out (Jambor et al...favorite tips and tricks, leave a comment below. Happy imaging! References and Resources References Andrews...

...and deliver any cargo you fancy (size limits do apply, however.) Why use a virus? Viruses are essentially...low immunogenicity which makes them ideal for applications with whole organisms. Finally, viral delivery...delivery is typically straightforward across applications, while electroporation of a human, for example, isn... post. If a viral vector seems like the right approach for your experiment, you’ll need to decide exactly...retro viruses can accommodate a cargo up to approximately 8 kb, but viral packaging is less efficient ... In fact, AAV gene therapies have already been approved in the US for multiple diseases, including hemophilia... (~4.5kb From basic science to clinical applications, viruses are true bench to bedside tools. Keep...

...allow you to be flexible in your approach to affinity reagent applications. Whatever affinity reagents you...re used in a number of research and clinical applications. Most of the time, when someone speaks about...deeper penetration into a tissue, useful for applications like IHC, or be able to access an epitope folded...that would size-exclude an antibody. In imaging applications, smaller affinity reagents means higher resolution...antibody’s ‘Y’. They are often used in clinical applications. Fab fragments can be created in lab in situations...Shahied, 2004). They are often used for clinical applications, including imaging.  Diabodies are ~60 kDa proteins...antibodies are still an excellent option for many applications, and frankly, if ain't broke, don't fix it!  ...