...potential applications. Challenges of existing mutagenesis methods Several continuous diversification methods... challenging for users to achieve targeted diversification of all nucleotides within a defined region ...Chang Lab have created methods that introduce diversification by targeting a site of interest with dCas9 ...level of variation that can be introduced.   Diversification is limited due to the integration of oligonucleotide...increasing the standard mutation rate seen during DNA replication by 120-fold over background. Due to its modular...only ~2-fold over nCas9 levels. Two sets of modifications were also made to PolI3M to increase the mutation...more than one mutation near the target site. Applications of EvolvR The Dueber Lab used EvolvR to discover...

...the optimal location. It is also important to have good information on the exact location of the TSS. ...appreciable levels of modification. Whole-genome sequencing of cells modified by CRISPR indicates that the consequences...specific base edit, or modulation of gene expression. Location and sequence are important considerations for ... gRNAs. For indels, it's not so important what location in the gene you target, but it is important that...fewer sequences to choose from). Finally, for HDR, location is much more important because you have to target...target site is far more constrained by the desired location of edit; efficiency decreases dramatically when...alternative to HDR for introducing edits. The same locational constraints are even more exquisite for the so-called...

...able to replicate, and for a virus to cross a synapse from one cell to another, it must replicate. Modified...like how our stem cells are housed in specific locations and give rise to progenitors during growth and...infects one cell (the “starter cell”). It then replicates within that cell, and nascent RABV cross synapses...infection occurs, subsequent cycles of viral replication and retrograde transmission occur, spreading ...limitations. First, these high levels of viral replication are toxic. Second, infecting subsequently higher...spread monosynaptically from these cells. These modifications take advantage of the fact that RABV is an enveloped...G-deleted RABV will infect the starter cell. RABV replication will then occur and the G protein (supplied in...

...species can be indicated in the “Species Other” column. Relevant Mutations Indicate the amino acid...usually very easy — our tech transfer team will communicate with your organization’s technology transfer ...name and match the name as it appears in its publication or as it is commonly known in your lab. Plasmid...descriptive purpose focuses on the practical application of the plasmid and will help a scientist outside...reagent/antibody, unspecified, or other. If other, indicate type in “Vector Type Other” column. For example...or room temperature. Growth Strain Please indicate which growth strain Addgene should propagate your...For plasmids that contain the R6Kγ origin of replication, choose the Pir1 strain. If your plasmid cannot...

...your experiment (all well validated for your application, of course!) When the stars are aligned and the...factors that affect isotype choice. Target and application should be considered The first question you may... a strong signal and to work in a variety of applications. The desired specificity of your secondary antibody...affect my experiment?” Well, it depends on the application and target. If you are running a basic western...target is expressed at very low levels or your application requires a high degree of affinity, then species...considered when choosing an antibody. In some applications probing species-on-species, i.e. using a mouse...mouse IgG2a and rat IgG1 antibodies in the same application, using a Goat anti-mouse IgG2a cross-adsorbed...

... utilize an array of tools that rely on DNA amplification and manipulation to produce and edit plasmids...Potential points where DNA errors can be introduced, indicated by exclamation marks. Error points include places...places where DNA is being created, such as PCR amplification or bacterial growth. Created with BioRender....through the QC pipeline to resolve any issues.  Catching and addressing QC issues Addgene’s QC team has...has developed a process for identifying, categorizing, and resolving a variety of QC issues. This process...discrepancies can be caught prior to distribution. Catching these errors prevents future experimental issues...during analysis. These include topics such as: locations in a plasmid that are less likely to affect function...

... series.  “The only healthy communication style is assertive communication.” - Jim Rohn, Author, Entrepreneur...I heard about supervisors in all of the above categories. In addition, I also learned about two other ...Assertiveness? An assertive person is able to communicate their ideas confidently while also considering...of the most frequent sources of conflict is miscommunication. For example, you might misunderstand your...the wrong direction. The best way to avoid miscommunication is to follow up after every meeting with an...workplace, these strategies will help you to communicate with confidence earn respect from your supervisor...in an email, there is a high likelihood of miscommunication, which can lead to even more conflicts. If...

...are, you’ll even have DNA left over for other applications, like PCR, cloning, transfection, or long-term...wash step or use a different strain. For most applications, Addgene uses and recommends DH5𝝰! This strain...cloning Bacterial strains adapted for protein purification use the cell’s resources to produce high levels...retransforming the plasmid into a strain meant for DNA purification. It sounds like extra work, but it will likely...your E. coli to ensure it is suitable for your application. We have previously reviewed many common lab ...partially specified by a plasmid’s origin of replication, but can also be affected by the plasmid size...the material used for inoculation. To further complicate matters, plasmid copy numbers are often ambiguous...

...need? Multiple first or corresponding author publications (published, not in preparation) A history of...The other items will follow. If you get more publications, you will be more competitive for fellowships...to write as well as mentor. Yet, even the most dedicated mentor is not always available for mentees due... project. Furthermore, Group Leader position applications require 3 letters of reference- the graduate...interests, complementary perspectives, a shared communication style. You “get” each other. The other factor...makes time for you, as needed, this is a strong indicator that this person cares about your science and ...spurious yet intriguing findings that fail to replicate. Plan experiments logically and strategically ...

...and GFP-edited amplification products (BRAF-GFP duplex) and from GFP-edited amplification products (GFP-GFP... wanted to inactivate the gene BRAF (a kinase implicated in several human cancers) in A549 cells (a human...transduced with Cas9 expressing lentivirus at the indicated multiplicity of infection (MOI). Anti-vinculin...need to use FACS (like they did in the original publication). I thought I could see GFP expression with a...expression, but I didn’t. 2. In the original publication they see GFP expression in about 20% of their...an MOI of 0.3, as recommended by the original publication. Pro-Tip: 1. Use multiple gRNAs to target different...the 428 bp Surveyor nuclease cleavage product indicates a gene edit. GFP gRNA-transduced Cas9-A549 cells...

...intended application - Remember, just because your antibody performs as expected in one application does ...strategies for antibody validation in different applications. Here, I want to highlight the general suggestions...intentionally generalizable and not specific to certain applications or antibody targets. That said, not every strategy...will be suitable for every antibody or every application. The goal is to show that your antibody has been...strategy to use will depend on your intended application and available resources.     Figure 1...confident in your data. This is a good option for applications like immunohistochemistry on human tissues, ... for validating antibodies for immunocapture applications and is amenable to high-throughput analyses,...

...and Jinfang Zhu. "Immunologic applications of conditional gene modification technology in the mouse." Current...contributed by Alessia Armezzani, scientific communication manager at genOway. A few decades ago, the brain...École Normale Supérieure de Lyon. Her love for educational science led her to pursue a teaching career, ..., and she now is a very happy scientific communication manager at genOway.     Acknowledgement:Through...Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein...): 7025-7030. PubMed PMID: 18614669. Belzung, Catherine, Marc Turiault, and Guy Griebel. "Optogenetics...Yizhar, and Karl Deisseroth. "The development and application of optogenetics." Annual review of neuroscience...

...light has limited applications in vivo. UV and blue light is readily absorbed and scattered by the skin, so...strategies Publication System nickname Photoactivatable moiety Cas9 variatn CRISPR protein application Split...improvements to CRISPR technology since its initial applications for genome engineering in 2012.  Many of these...scientists to use light to externally control the location, timing, and reversibility of the genome editing...enzymatic activity. Photoactivatable genome modifications by NHEJ and HDR using split systems Later in...mutations via NHEJ (frequency of 20.5%) and induced modifications by HDR (frequency of 7.2%). The authors additionally...light (730 nm). This nucleotide triggers the translocation of the protein BldD to the nucleus. To couple...

...insert will typically contain both a protein modification sequence and a selectable gene product for isolation...tags have been integrated. Simply design your amplification primers with the desired targeting homology—.... cerevisiae, looking at their performance in categories such as brightness, stability, and disruption...optimal FP combinations for use in yeast imaging, categorized by specific filter sets and experimental output...This collection is ideal for tandem affinity purification of protein complexes. John Pringle and Jürg ...deposited a large collection of plasmids for genome modification in yeast developed by Dr. Pringle’s former lab...collection of plasmids for a wide variety of genome modifications in S. Pombe, including full and partial gene...

...take to achieve a clean NTC: Have a dedicated bench with a dedicated set of pipettes for ddPCR set-up. .... Each droplet contains the ingredients for amplification of the target DNA. This partitioning reduces...detection of the product. As an added benefit, replicates are built into the technology. One well (containing...droplet contains the material required for a mini amplification reaction to take place.  When the PCR is complete...number of cycles. After additional rounds of amplification, your fluorescence amplitude will increase, ... use ddPCR for AAV titration, there are many applications for the technology.  Droplet digital PCR is ...being used in the detection of microbes. Some applications include measuring viable probiotics and detecting...

... put it best when he stated, “the value of an education . . . is not the learning of many facts but the...possible to bring the kinds of transformative educational experiences afforded to those fortunate enough...which can lead to greater success in meeting educational goals (Kowalski et al., 2016; Scott, 2021). In...CUREs are beginning to transform undergraduate education in the sciences by making a greater number of ...scientific research more inclusive. CBE Life Sciences Education. 13(4):602-606. Brian, D. 1996, Einstein: A Life...positive STEM attitudes in CUREs. CBE Life Sciences Education. 21(4):ar74. Duboue, E., Kowalko, J., Keene, A...biochemistry laboratory: Part I – guided inquiry – purification and characterization of a fusion protein: Histidine...

...caudal NST. (A-C) Low-magnification images showing viral spread. (D-F)  High-magnification images immuno-stained... right AAV for your system or to successfully replicate published works. Of particular importance to these...improve our Addgene Data Hub, 76% of respondents indicated they wanted to see more negative data (Figure ...minimum n = 2, and well labeled high- and low-magnification images demonstrating vector performance) will...performance. Yet, most of the technical data needed to replicate and build on others’ results is available not ...information provided in openly accessible forms like publications. Why not get this data out there?  The Addgene...Sommer, J. M., Pierce, G. F., & Qu, G. (2005). Identification of factors that contribute to recombinant AAV2...

...for both transient and stable gene expression applications in plants. This smaller, easily customizable...Agrobacterium are termed binary vectors because they must replicate in two species: Agrobacterium and E. coli. Like.... coli. However, they also need to be able to replicate in Agrobacterium to ensure that they are maintained... Thus, binary vectors contain two origins of replication, one each for E. coli and Agrobacterium, along...bases each. By switching the E. coli origin of replication and codon-optimizing the Agrobacterium ORI, they...constitutively active M24 promoter has been modified with duplicated enhancer domains and characterized in studies...these properties of pSiM24 are suitable for many applications, the plasmid can also be customized due to its...

...software that let me create graphics that better communicated the science in my blog posts. This post highlights...free tools which will hopefully also help you communicate your science, whether it’s in presentations, ...number of drawing tools, so it’s harder to draw intricate figures, like a brain or a mouse. Vectr Vectr ...without the watermark, which are better suited for publications, if you sign up for a paid account.  I’m proud... free medical images organized into four main categories: anatomy and the human body, cellular biology...appropriate credit, provide a link to the license, and indicate if changes were made to the images. This requirement...easier to give attribution, than for journal publications. Google Image Search Google image search is ...

...Rep78/68 play a key role in AAV genome replication. Replication initiates from the AAV genome ITRs on ...contains the proteins needed for the virus to replicate,  and the transfer plasmid which contains the ...aka the RepCap plasmid encodes two genes: the replication (rep) gene and the capsid (cap) gene. In wild-type...its products. Created with BioRender.com   Replication (rep) gene The rep gene encodes four proteins...proteins that are required for viral genome replication and packaging: Rep78, Rep68, Rep52, and Rep40. Rep gene... generate a 3’ hydroxyl group needed for DNA replication, Rep78/68 binds the Rep binding element (RBE)...nicked by its endonuclease domain. Following replication, Rep78/68 stays attached to the AAV genome and...