...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 ...

...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...

...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...

...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!  ...

... chain reaction (LCR)-overview and applications." PCR Methods Appl 3.4 (1994): S51-64. PubMed PMID: 8173509...or DNA base pair mismatches (8-11). For some applications, there cannot be any end-joining activity, and...regions. Why use a high fidelity ligase? Numerous applications have been developed to take advantage of the...Taq and other thermostable DNA ligases. These applications detect specific nucleotide sequences with high...Ligase are not replacements for T4 DNA Ligase in applications such as routine cloning or DNA library preparation...Temperature Calculator can be used to estimate an appropriate incubation temperature when using NEB thermostable...Christopher D. Lima. "The polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases...

...sequencing is laborious, time consuming and cannot be applied to heterogenous populations (Bell et al. 2014).... on genome sequencing. Used in concert, these approaches can provide the researcher with a reasonable ...variable) Sequence-level data Low-throughput, inappropriate for heterogenous populations, low sensitivity...(Fu et al. 2013; Pattanayak et al. 2013). This approach misses larger mismatches (e.g. six nucleotides...Genotyping Options Technique Detection Limit Application Advantages Disadvantages GUIDE-Seq 0.1% Cell-based... may be to integrate both biased and unbiased approaches. As suggested in a review by Tycko et al., 2016...a highly accurate manner. Using both of these approaches may not be necessary for every CRISPR experiment...

...gRNA targeting approach is then used to direct the complex to a specific locus. This approach provides a ...tools available for bacteria and some of the applications for which they’ve been used. The beginnings ... E. coli. Furthermore, to demonstrate wider applicability they demonstrated success of Cas9 mediated repression...method in E. coli, Jesse Zalatan’s lab used an approach to identify known proteins involved in the activation...scenario allows for several potential CRISPR applications. First, CRISPR can be used to target locations...resolve them, and the cells die.  This could be applied to diseases where the presence of just a single...polymorphisms, including antibiotic resistance genes. This approach successfully targeted specific organisms when ...

...need for low-cost, field-based diagnostics, our approach could make an impact. Our motivation was underscored...involved in the effort. This inclusive and open approach by all involved definitely contributed to our ...). Using toehold switch-based RNA sensors, our approach uses portions of the pathogen’s genome as unique...portable quantification of diagnostic results.  Our approach now detects samples at clinically relevant concentrations...longer-term aim is to advance our programmable approach to other critical needs and to further develop...focused on developing cell-free synthetic biology applications for human health and the environment.     Alexander...nanomaterials science, and self-assembly for applications in health, energy, and sustainability.   References...

... necessary to apply a compensation value between the two channels (Figure 1A). Applying compensation means...your single-color controls as a starting point. Applying compensation to your experimental samples In order... in the plot. A compensation value of five was applied to APC-Cy7 against APC to correct for this bleed...compensation value of 3.7 for APC into APC-Cy7 applied next, which fixed the bleed in this population....each plot.  The general rules of compensation apply to your experimental samples as they did with your...populations and may need to be “comped” first before you apply compensation values to your current channels. A ...plot, and the population does not move even after applying very large compensation values (100+), consider...