...missed with conventional FP imaging. PA-FPs are divided into 2 main categories - those with irreversible...requires a temperature below 30°C and this is not ideal for experiments in mammalian cells. Dendra2 is another...light both activates and inactivates Dronpa is not ideal, since this means Dronpa is quickly inactivated ...your experiment, there are several parameters to consider, such as brightness, photostability, pH stability...protein aggregation. Another important parameter to consider is the contrast ratio- a comparison of PA-FP brightness...intensity used to activate the PA-FP should also be considered. If the light intensity needed for activation...to preserve cellular structures (0.5-1% osmium tetroxide). These conditions destroy most PA-FPs, but, in...

...Is the conference you are attending on the larger side? At large conferences, chances are that several ...have additional questions. If you or your lab identifies a potential collaborator from the meeting, reach...If your poster material is nearing publication, consider uploading your work to a preprint server (and ...Perhaps some of your poster visitors had a great idea or interesting questions worth discussing with your...Reach out if you’d like to meet someone and set aside a time to meet. Some conferences may also have a... of the things we've mentioned above could be considered networking, but here are some more general tips...resume and prepare business cards. You might not consider being a student or postdoc a “business,” but having...

...goal was to enter industry since I was drawn to the idea of working as a team to develop therapies that could... of academics tend to view industry as “the dark side,” that you’ve sold out. You’ve given up your intellectual...ve discovered.) As someone who has now seen both sides of the postdoc experience, I’m going to share what...an industry postdoc is something that you’re considering. Like anything, there are pros and cons to postdocs...will hire a group of postdocs and then let them decide what they’d like to work on later. For instance...suit your needs better than others. Whether you decide to do an industry or an academic postdoc, you’ll... open positions at Addgene Check out our career videos ...

...treatment or prophylaxis of COVID-19. Researchers identified nanobodies that bind to coronavirus spike proteins... randomized with a defined set of different trinucleotides (corresponding to different amino acids) in...bind to the antigen. The Seeger lab can directly provide the three sybody libraries in the form of mRNA ...tags are used in a final enrichment step for identifying sybodies that bind strongly and specifically ...their protocol because it is less susceptible to identifying unspecific and low-affinity sybodies (Zimmermann...-30) of sybody candidates are sequenced for identification. All unique sybodies can be subcloned into ...demonstrated they could use the protocol to rapidly identify and isolate sybodies targeting the SARS-CoV-2 ...

...-Cy7 and PE-Cy7; or BV421 and BV51. It is a good idea to look up new fluorescent markers before deciding...them, using a tool like BioLegend’s Fluorophore Guide Chart.  Pro tip! Some antibodies go by different...setup. For instance, BV510 and AmCyan have almost identical emission spectra but may be called BV510 on one...which way your population needs to be shifted, consider your perpendicular lines. In this case, the population...APC-Cy7 are both bleeding into each other, as is evident by the two diagonal populations in the plot. A ...applying very large compensation values (100+), consider what two markers your fluorescent markers are ...fluorescent markers can be challenging! But we hope this guide can help you get started. Remember, you can always...

...such a system is likely to introduce additional nucleotides flanking the insertion. If the DNA ends anneal...MMEJ may also introduce, substitute, or delete nucleotides in addition to the expected deletion, but the...breaks are necessary for knock-in: one on either side of the GFP-Puro cassette and one in between the ... last coding exon of your gene of interest. For ideal use, this gRNA should be cloned into a vector containing...cuts in a staggered pattern, it is thought to be ideal for HDR-independent knock-ins, but this possibility...Engineering Tools like Sleeping Beauty Mutase Find the Ideal CRISPR Software for Your Experiment Additional ...Addgene.org Learn All about CRISPR on Our CRISPR Guide Pages Find CRISPR Plasmids for Your Experiments ...

...you reach for that silica spin column, stop to consider some ways to purify DNA without a kit. DNA purification... kits have advantages: they are convenient and provide uniform, consistent results. But they are also ...method described in Kim and Morrison is nearly identical to that of commercially available kits but instead...concentration of a chaotropic salt (guanidine hydrochloride for the plasmid purification protocol and guanidine... Image: Kim and Morrison, 2009. This method provides DNA yield and quality similar to that obtained.... Homemade buffers performed the same as those provided with the kit. Although not tested by the authors... Molecular Biology Reference Visit our protocol video library References Kim, Y., & Morrison, S.L. (2009...

...Working with AAV vectors in neuroscience Let’s consider my first challenge when I started working with...). However, this one set of experiments didn’t provide any information on other important factors such... science initiatives, like Addgene’s Data Hub, provide an avenue to more easily solve these frustrating...every researcher experiences and that it often provides incredibly valuable insight into one’s work. As...different methods and vectors, and use this data to guide them in their own experiments. By providing DOIs...highlights the dearth of AAV vector information provided in openly accessible forms like publications. ...Sommer, J. M., Pierce, G. F., & Qu, G. (2005). Identification of factors that contribute to recombinant AAV2...

...biological disciplines and scientists employ GFP for a wide number of functions, including: tagging genes for... in the wildtype protein and facilitating its widespread use in research. Many other mutations have since... that many fluorescent proteins found on the red side of the spectrum are not GFP derivatives, but are...folding, maturation, and fluorescence. Biosensors: A wide array of GFP-based fluorescent biosensors has been...plasmids often include GFP as a marker to help identify which cells have successfully taken up the plasmid...applications for GFP. It has also been used to identify particular cell populations in drug screens, to...Plasmids 101 Articles Addgene's Fluorescent Protein Guide Note: A. Max Juchheim contributed to the writing...

...around $45,000, but as I’ve discussed elsewhere (slides here) there are many postdocs paid at much lower...must be met. In the Department of Labor’s general guide to the effect on higher education, you can see that...and Brandeis universities have stated that they consider postdocs paid on stipends to be FLSA exempt, and...institutions; and that institutions which can more easily provide bridge funding to absorb the cost will encounter...jobs that the majority of postdocs end up getting outside academia. There is also a valuable lesson for the...level they will be raised to on Dec 1st - across a wide consensus of academic stakeholders. There was ample...going away, and it is becoming ever clearer that outside scrutiny of science and the academic system is ...

...site, and titer. Addgene’s Viral Vector Service Besides being complex to use, viral vectors are also difficult... bias: Addgene uses a prioritization process to decide which tools we make available. This prioritization...community. It does, however, necessitate us first identifying broadly useful vectors, a process that isn’t ...information from requestors and depositors, and identifying trends in our own repository data, giving us ...viral vector educational resources, from eBooks to guides to protocols, which was why I was so excited to...2023; Haery et al., 2019; McDade et al., 2016), video, educational resources, and collaborations, and ...Swanson, L. E., & Fan, M. (2016). Practical Considerations for Using Pooled Lentiviral CRISPR Libraries...

...letters, while higher-level jobs are more likely to consider cover letters an important part of the application... content team, where writing is a core skill, considers cover letters to be very important. On the other...part of the job application process, it's a good idea to talk to people in the field to understand what...cover letter for? A cover letter is a place to provide helpful context about your candidacy, beyond what...make it easy to master ddPCR.  Finally, it’s a good idea to state why you’re applying to the specific company...unofficial poll, hiring managers also said that they considered cover letters to be more important for higher-level...Additional Resources Hamilton College Cover Letter Guide How to Write a Cover Letter Blog Post from Hubspot...

...hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com. Inosine...act on newly-deaminated inosines. Pro tip! The nucleoside inosine consists of the nucleobase hypoxanthine...transversion editor AYBEv3. This tool still retained considerable A → G editing activity at most sites, in addition...initial editor AXBE, for A → X base editor (X = any nucleotide). To create a more specific A → C base editor...be useful for saturation mutagenesis, and they provide a strong proof of concept for further development...efficiency, purity, and indel rate of each tool vary considerably. And there’s so much more exciting protein engineering...base editing Get a broad overview with our CRISPR Guide Check out our CRISPR 101 ebook ...

...invariably inconclusive and unreliable. Proper controls provide the constant variables that enable the correct ...functionality of your experimental system and help identify opportunities for troubleshooting or optimization...experiment, untreated cells should be included as these provide the baseline/standard against which other samples...correct interpretation of the results because it provides an important reference point when analyzing the... but the papers cited in the reference section provide more in depth information. Technical replicates... time consuming and ultimately more important. Ideally, each biological replicate should use fresh media...plasmids, be performed on different days, etc, but outside constraints may impose some limitations and you...

... of multiple animals on different occasions via video, generating huge amounts of data. When published...experiment would require access to each individual video. To PLOS’s credit, the organization subsequently...subsequently clarified what the policy intent was and provided an FAQ list to further explain the policy. The fact...variation is calculated and placed in the legend. Widely reported difficulties in reproducing experimental...the author or their host institution expected to provide data in formats exported to common file types? ...could be mitigated by on-line services such as SlideShare, Figshare and Dataverse. Just as Addgene helps...than just a single projection of it, could allow idea generation and extension, based on existing, published...

...with them. If you decided to make your own competent cells using the protocols provided above, then you ...cells are available online. A few examples are provided here for reference: OpenWetWare Preparing chemically...phase, then wash and resuspend the cells in the provided buffers. The competent cells are then aliquoted...Stbl3TM. How to transform your plasmid DNA If you decided to use commercial competent cells, then you're ...transforming your cells? Check out our transformation guide.   ...

...mutations found in leukemia, the Lowe lab was able to identify a set of mutations that made cancer cells more...mouse line. Over the years the fly has become an ideal model organism to study an array of topics including... to create transgenic animals. Zebrafish are an ideal model organisms for embryonic development studies... genes have an ortholog in C. elegans making it ideal to study basic molecular biology processes. C. elegans...quiescent and proliferative pancreatic β-cells identified by HOTcre mediated labeling." Proceedings of ...Menezes, Regina, et al. "From the baker to the bedside: yeast models of Parkinson's disease." Microbial...Additional resources on the Addgene blog Read our quick guide to Drosophila Download the fluorescent proteins ...

...Addgene’s catalog is a new class of sensors for neuropeptides and neurotransmitters. Motivated to develop ...GPCRs recognizing various neurotransmitters, neuropeptides and other ligands - almost A to Z! - from ATP...vectors (here and here).  Figure 3: JEDI-2P was identified from a high-throughput screen, selected for increased...existing sensors. Using genome mining and structure-guided engineering of potassium binding proteins, they...point for further engineering. Figure 5: Structure-guided design and genome mining both led to new sensors...Fluorescent Potassium Indicators through Structure-Guided and Genome Mining Strategies. ACS Sens. 2022 May...protein plasmids and resources Fluorescent protein guide: Biosensors Collection ...

...loading control: the intensity of each POI band is divided by the intensity of the loading control in the ...problems for proteins of very different abundance, consider the following example gel. Figure 5: An...intensity axis. But the loading control points are outside of the linear detection range. As a result, they... problematic for computational quantification. Outside the linear detection range, band intensity measurements...experimental conditions. Otherwise, you might want to consider a different loading control. Assumption #2: The...final blot. Quantifying the entire lane of protein provides a loading control that can be used for normalization...The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Review...

...New ideas and approaches are encouraged in science. It isn’t surprising that new ideas are also popping...before you even receive the product? How can we consider the environmental impact of those processes? Who...might sound risky in terms of contamination, but consider flow cytometers. These fluidic machines handle...through an EPA-approved process and no longer considered hazardous. Then, they use this material to produce...plastic lumber and distribute it to industrial and residential markets. Rethinking our approach to lab waste...