...International Publishing. https://doi.org/10.1007/978-3-319-07758-1_2 More resources on the Addgene blog Viral... using biorender.com   If you’re new to talking about viruses, here’s a quick tip on terminology. “Virus...have a growing number of Viral Vectors 101 posts about each type, so be sure to check our blog, or our ...

...fluorophore's excitation spectra around 640 nm (Figure 3), we see that APC-Cy7 is excited at a lower efficiency...cannot be excited at this wavelength. Figure 3: Excitation and emission spectra for APC and APC-Cy7...spectrum is located in a range of wavelengths of about 380–700 nm. Photons of lower wavelengths are higher... 650 nm, APC-Cy7's global excitation maximum is about 750 nm. If we were to use a laser that excites at...optimal excitation happens at higher wavelengths of about 750 nm. Source: https://www.bdbiosciences.com/en-us... we start building your first panel, we'll talk about one last principle of color emission. As you can...our blog post on color compensation to learn more about them. Alright, let's build your first panel! Designing...

...Reference Rq(R165L) Human M3 muscarinic Arrestin-2/-3 CNO* Increase Arrestin translocation Arrestin signalling...activity. PSAM-5HT3 LGICs pair a PSAM domain with a 5-HT 3 serotonin receptor sodium- or potassium-selective ...K + ions, and activates neuronal activity. Figure 3: PSAM4-based ligand-gated ion channels, their effect...routes. Image courtesy of Ute Hochgeschwender . Table 3. Descriptions of LMOs and their activity in neurons.... However, CNO is back-metabolized to clozapine and other clozapine metabolites and these can have off-target...concentrations of clozapine N ‐oxide (CNO). CNO is a metabolite of the antipsychotic clozapine and seems to be...experiments in this species may be entirely due to back-metabolism of CNO to clozapine, demonstrating the need for...

...three systems to analyze single cells from a mixture (3): An optics system A fluidics system An electronics...expression. A routine flow cytometry experiment uses 2 or 3 different fluorescent colors each measuring a different...biosafety cabinet with cloning rings or following labor-intensive limiting dilution protocols? If this sounds...fluorescent microscopy must be used. To learn more about the most suitable fluorescent microscopy technique...a laser and the instrument collects information about the cell’s size, complexity and fluorescence intensity...captured by detectors and provides useful information about the cell. Light that continues in the same direction... forward scatter (FSC) and provides information about the relative size of the cell; larger cells produce...

...preparation, and lead your own research project.  3. Do you prefer being a generalist? Working on a variety... Resources Resources on the Addgene blog Learn about career planning resources What Do I Do Now? Academic...

...Palindromic Repeats, in Pubmed returned 728 articles (3/12/2014). With so many options for CRISPR plasmid ... gRNA design tools Need more questions answered about CRISPRs? Check out the full list of 16 FAQs answered...

...identifier for individual cells. Each cell is labeled with 3-4 tags to increase the diversity of the CellTag library...Additional resources on the Addgene blog Learn more about induced pluripotent stems cells (iPSCS) and reprogramming...

...(i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy techniques   Fluorescent...methods 2, 920-931 (2005). PubMed PMID: 16299477. 3. Eggeling C, Willig KI, Sahl SJ, Hell SW. Lens-based...call Epi-fluorescence microscopes. (See left image above for an example image). Point Scanning Confocal Microscopy... plane to reach the detector and all light from above or below is excluded. Parallelized Confocal Microscopy...due to limitations in light penetration mentioned above (5). Thick static samples Ex: Fixed tissue sections...sectioning. Point scanning confocal microscopes (see above) often provide the highest quality images for this...

...tagged with different fluorescent proteins or dyes. 3. Two-photon microscopy Two-photon excitation microscopy...PMID: 18718768. PubMed Central PMCID: PMC2569002. 3. Egen, Jackson G., et al. "Macrophage and T cell dynamics...fluorescence microscope where the light source is mounted above (epi) the specimen and the excitation light passes...technique that allows imaging of living tissue up to about one millimeter in depth. Two low-energy photons ...response, but the study of these dynamics was still laborious as epifluorescence microscopy wasn’t able to give...skin. Two-photon intravital microscopy. The video above depicts the infiltration of neutrophils expressing...

...strand completely; it is essential, however, that the 3’ end of the primer corresponds completely to the template...proceed. Usually a guanine or cytosine is used at the 3’ end, and the 5’ end of the primer usually has stretches...stretches of several nucleotides. Also, both of the 3’ ends of the hybridized primers must point toward ...restriction site at the 5’ end of your primer, note that a 3-6 base pair "clamp" should be added upstream in order...amplification process. When designing, if unsure about what nucleotide to put at a certain position within...capabilities. Taking into consideration the information above, primers should generally have the following properties...

...-mediated marker excision, it takes approximately 3-4 weeks to obtain a homozygous, outcrossed knock-in...produced a higher editing efficiency, while for edits 3’ to the PAM sequences corresponding to the opposite...learn more about genome editing with CRISPRs? Check-out Addgene's CRISPR Resources Learn about gRNA design...interest, I encountered low efficiency (0.13%) and laborious handling (screening 768 F1 animals). Recovering...

...assist with restriction enzyme digestion (usually 3-6bp). Restriction Site: Your chosen restriction site...Primer will use the sequence 5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds the ORF and we will add...our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC-3'. Many restriction enzymes do not cut DNA efficiently...more information). Thus, we recommend that you add 3-6 bases upstream of your restriction site to improve... sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design is similar, but...including the stop codon (5'-TGGCATATCTCGAAGTACTGA-3'), then adding NotI (GCGGCCGC) and then TAAGCA to ...sequence of 5'-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3' (30bp with 18bp of homology to the ORF). We now ...

...one company, likely will need to move around every 3-6 years—a short and exciting ride Complicated matrix...students Collaborations may be less “complicated” - for example, easy to initiate a collaboration without...up the pace Constant worry about grants and renewals What is different about non-academia? Scientific...make a decision about something like this without sufficient data? Starting to learn about the diverse options...with scientists just like you What is different about academia? Publishing in peer reviewed journals ...without having to worry about proprietary information Work hours are long, but there is more flexibility with...paths to pursue. Spend some time really thinking about your motivations and what parts of being a scientist...

...and New Faculty" downloadable book. Skip to Chapter 3 for tips on how to develop your role as a lab leader...start their own academic research laboratory. How does one even go about starting a lab? Obviously, funding...always helps. But how does one build a strong collaborative research team motivated by similar visions and...booklet. What has been the most surprising thing about becoming a PI? Tom Ellis has a one word answer: ... has to deal with. My inbox currently stands at about 65,000 messages and dealing with email can be half...the first couple of hires to build a strong, collaborative team. And take advantage of the many resources...Medical School) for taking the time to speak with us about adding new scientists to their labs. Further Reading...

...ready for the live audiences in your future.     3. Start something In almost every career talk I present...mentoring group (see above). However, now that we are all so good with video calls, how about trying your hand...blog or podcast, initiated and maintained a collaborative project, served on the board of a professional...appointments with people all over the world. Think about contacting alumni of your lab or university or use...suggest you speak with. See this post that’s all about informational interviews, for more details on how...progress on both building relationships and thinking about future career options. 5. Write a grant - even if...

...and (3) development of tools for data analysis/visualization using R/Shiny. She is passionate about open...section-specific comments, I re-read what I wrote about the paper after my initial read, adjust my language...wish for The last thing that I wanted to mention about the peer-review, is that as a peer-reviewer, you...each peer-review, I try to mention what I liked about this paper and recognize the hard work that the ...review article Resources on Addgene.org Learn about depositing plasmids pre-publication Find molecular...

... of the Brunello library are also shown in Figure 3. I hope you’ll agree with me that they look very similar... and we thought it would be a good time to talk about the amplification and verification processes we ... DNA. These reactions should allow us to obtain about 100 to 1,000 bacterial colonies per individual plasmid...from the count_spacers.py file from Feng Zhang’s laboratory. There are a number of other analysis scripts...you have any tips, input, or stories of interest about library use, let us know in the comments! Additional...NGS Sequencing to Validate AAV Preps Learn More about Pooled CRISPR Libraries Resources on Addgene.org...

...Brandon Sanderson "The Mistborn series is the first 3 books of a larger epic of fantasy novels. Set in a...resources on the Addgene blog Learn about company culture at Addgene Read about other lunch activities at Addgene...scientists bring biases to research and don't collaborate with others to bring diverse perspectives. Everyone...Science is a terrifying and equally good wake up call about the science of race."     - Joanne Kamens, Executive...easy reads, some are hard. Some I’ll never think about again, some I loved so much I’ll force them on everyone...

...page. Figure 7: Enter information about the gene/insert(s). Step 3: Cloning Information Please enter ... of 10 or more plasmids.       Figure 3: If submitting plasmid(s) online (left), choose to...

...et al., 2014) (Addgene #104106 - #104108) (Figure 3) that can be introduced using in utero electroporation...Additional resources on the Addgene blog Read about Brainbow Learn about using AAVs for neuronal tracing Visit...page Additional resources on Addgene.org Learn about Addgene's viral service See our fluorescent protein...

...stressed or aging cell populations. Figure 3: Micrographs of wild type (top panels) and cells with... plasmids you need. If you'd ever like to write about a recent plasmid deposit please sign up here.  ...non-overlapping cells. Until now, tools for doing this were labor-intensive and required using manipulations such ...

...thousands of loops. These loops not only provide a 3-D structure for packing genomic DNA into nuclei, but... plasmids you need. If you'd ever like to write about a recent plasmid deposit please sign up here.    ...neurons. Earlier this year, the Hegemann lab and collaborators report their discovery of a new family of seven...

...Lebbink RJ, Rehmann H, Geijsen N. Cell. 2015 Apr 23;161(3):674-690. doi:10.1016/j.cell.2015.03.028. PubMed. ... a protocol has been established) may be less laborious than viral purification, and it does not raise...

...initiate. The TATA box is a DNA sequence (5'-TATAAA-3') within the core promoter region where general transcription...promote the initiation of transcription. The most 3' portion (closest to the gene's start codon) of the...of interest is expressed. Summary Promoters are about 100-1000 base pairs long and are adjacent and typically...Inducible by arabinose Promoter of the arabinose metabolic operon trp Repressible by tryptophan Promoter ...

.../ I203R) (2) and PA-mRFP1(S146H / I161V / I197H) (3) were derived from DsRed and mRFP, respectively. When...PubMed PMID:19169259. PubMed Central PMCID:PMC2901231. 3.Verkhusha, Vladislav V., and Alexander Sorkin. "Conversion...emitted, making it easier to capture a clear image above background. The majority of PA-FPs come in 2 flavours...Additional Resources on the Addgene Blog Read More about Fluorescent Protein Imaging Choosing Your Fluorescent...

...because it required only about 15 minutes of my hands on time and a total of about 3 hours. It was robust,...Addgene lab. In this post, I’ll tell you a little bit about what I learned from a New England Biolabs (NEB) ...this, but thought we could learn a little bit more about high throughput DNA prepping by trying out NEB’s... new teaching space at Lab Central to learn all about it. LabCentral is a lab and office space shared ...without breaking their bank accounts. When I heard about how LabCentral aims to enable emerging biotech companies...different biotech companies to interact and learn more about one another. They also provided both breakfast, ...go to a workshop because it will help you think about the most useful pieces of information from the workshop...

... motivated reasoning. Psychological bulletin, 108(3), 480. More resources on the Addgene blog Summer SciComm...discussions about the pros and cons of different fabrics while we worked. We also had discussions about the difference...grad student. I chose to focus on misinformation about sexual and reproductive health, as the stigma around... misinformation can flourish. I’ve never forgot about all the anxious, whispered questions from friends...the study design or by their preconceived ideas about the controversial medical issues. Participants ...none of the participants had preconceived opinions about “adeolitis.” The participants in my study were ...make reusable cloth menstrual pads while we talked about the environmental impact of disposable products....

... scientists have certainly not lost site of that. 3. Stem cell science is hard, and expensive Dr. Yamanaka...the Addgene Blog Learn about Efforts to Fight Rare disease with iPSCs Read About Addgene's Outreach Efforts...the first clinical trial with iPSCs with his collaborator Takahashi in 2014 – just 8 years after the OSKM...researchers special: 1. Stem cell scientists care deeply about ethics Ethics must play a prominent role in the ...Researcher Darrell Kotton 2. Stem cell scientists care about patients As any grad student who has spent years... a big picture perspective: Dr. Yamanaka talked about his clinical trial patients with advanced macular...proof that these researchers are thriving in a collaborative, rather than a combative, field. Of course they...

...the filters on the right to find single, dual, and 3+ cutters. You can also sort enzymes by clicking on...see the new map in action). Read on to learn more about the improvements we’ve implemented and how they’...more functional display, you can start thinking about your next cloning experiment early on. Beyond these... to to download the static image file (PNG, see above) for your own reference or to paste directly into...identified a plasmid that you want to learn more about, updates to our sequence analyzer will allow you...the features shown in the static maps discussed above, but also have interactive functionalities. You ...enzyme, primer, or ORF to learn more information about it. For example, if you hover over the ampicillin...

...an updated protocol), send an email instead. The 3 A's - Agendas, Action items, and Accountability Agendas...each of your meeting types (see above). We’ve provided some tips above for what works well at Addgene,...opposite effect. A great meeting should produce collaboration - a sense of dialogue and community among participants...meeting to discuss something difficult, be excited about the prospect of working toward a solution. At the...sections for each person to add a bullet or two about what they are working on. Building this into the...purpose of the meeting (see “defining the scope” above) or if you can cut it down to thirty minutes. ...some tips. Before long, you will be running collaborative, informative, energizing meetings all your own...

...produce and distribute virus. Addgene is a 501(c)3 nonprofit and our plasmid service was started based on ...sponsored the capital construction of our new BL2+ laboratory and the infrastructure capabilities to produce...our generous, sharing depositors. Over 3,000 laboratories are sharing plasmids (with 16 labs currently...helping thousands of sharing scientists from laboratories all over the world. With this reach and exposure...In early 2016 we surveyed hundreds of scientists about various ways we might expand Addgene’s services....provides viral vector technical service (details about virus production protocols and quality control can...material. The Outreach Team is getting the word out about this new resource and providing fantastically useful...

...great tool for someone starting cell culture work. 3. NEB Tools by New England BioLabs In my lab we do ...saved and cannot be deleted, so there is no worry about misplacing your results or losing that lab notebook...enzyme, or name to discover all the information about that restriction enzyme or share it via email with... this app. However, if you get quizzed randomly about a hazardous chemical and can’t find your lab’s MSDS... new researchers or students beginning to learn about biochemistry. The least this app will do is allow...

...specifically expressing Cre in the hippocampus (Fig. 3). The Cre-dependent AAV would access the neurons (... Get Started with AAV Learn more about Lentivirus Find out More about Addgene's Viral Service Resources...We often think about the brain’s function in terms of its regions. But equally important is the way the...head bone’s connected to the neck bone,” but with about a hundred billion neurons and six hundred trillion...

...recombination. In most recombineering strategies, only 3-4 phage proteins are needed and these can be provided...from previously discussed methods. As described above, you design two primers, each containing the desired...suited for insertions and deletions. The figure above shows guidelines for primer design; a desired insertion...mutation-containing colonies/total colonies) is about 84% for each of the three methods. However, REPLACR-mutagenesis... Additional Resources on the Addgene Blog Read about Other Plasmid Cloning Techniques like SLIC and Golden...

... Obligate aerobe Number of chromosomes 16 3 4 Preferred carbon source Glucose Glucose...pombe have dominated the research scene. But what about the other yeasts – in the biomedical world, are ...pharmaceutical companies (along with some research laboratories) have picked up the system for some of the uses...eukaryotic cells and peroxisomes are where methanol metabolism occurs in methylotrophic organisms. K. phaffii...

... epitopes that will likely bring about unwanted immunogenicity. The 3-D models can be applied to corroborate...limitations. Using methods together can inform decisions about in vivo application and clinical testing. This would...to drugs: adverse reaction terminology. WHO Collaborating Centre for International Drug Monitoring, Uppsala...

...Cepko 83898 AAVrg pAAV-mDlx-ChR2-mCherry-Fishell-3 Gordon Fishell   Chemogenetics Chemogenetics offers...vector name and serotype you’d like to be notified about. Chemogenetics Plasmid Serotype Name 44362 ...vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols and...

... lab E. coli K12 strains such as DH5alpha contain 3 methylases that recognize and methylate different ...questions is the same--methylation! Read on to learn about how DNA methylation may affect your restriction ...be methylated. Using the same example plasmid as above (which has the XbaI site blocked by methylation)...

...development since its inaugural year in 2012. That’s just 3 to 4 years ago. It is a bubble of innovation (a piñata...PMID: 23873081. PubMed Central PMCID: PMC3969858. 3. Doench, John G., et al. "Rational design of highly...databases mentioned above or, even better, first-hand knowledge from laboratories employing CRISPR on ... CRISPR system was involved. Today, that same laboratory enjoys a successful edit rate of over 90% in ...targetable NGG PAM sites whereas the human genome has about 300 million. With such a sparsely targetable genome...worse by an ever increasing marketplace. Since 2013 about 11 tools have been added every year.   Breaking ...databases are still young and will require a collaborative effort from the community in order to succeed...

...mutations was surprising,” says Balazs.   Figure 3: Neutralization outcomes of convalescent sera with...Browse all COVID-19 related blog posts Learn more about pseudotyping Resources on Addgene.org Find plasmids...

...cytoplasmic (CKB1) RNA target. Adapted from Figure 3 Colognori et al 2022.   Find CRISPR Csm plasmids... minutes of FUS.  Similar constructs to the one above can be made for expressing your favorite T-cell ...

...). Cells should die within 3-5 days and resistant colonies appear in about 10-14 days depending on how...

...guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent motif (PAM) on the opposite...sites with A's, producing C → A edits. Figure 3: Mechanism of cytosine transversion editors. Red arrows...still isn’t over! Keep an eye out for our next post about base editing, where we’ll tackle transversion editors...CRISPR 101: Cytosine and Adenine Base Editors Read about four base editor reporters CRISPR 101: Prime Editing...

...Addgene's COVID-19 plasmids and resources! Case study 3: Mouse strains Reagents developed during the SARS-...the Addgene blog Read more about COVID-19 databases and resources Learn about Addgene's work to share plasmids... availability of this strain from The Jackson Laboratory. Originally developed and characterized by Stanley...mouse strain is regaining popularity. The Jackson Laboratory, a nonprofit research institution, has been able...clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study. ...

... The DNA packed into our chromosomes is very much 3-dimensional, as you can see by this post’s chromatin...Leah Bury of Microscopic Art. I started thinking about this problem when I recently attended the 2018 Chromosome...lot of scientists who not only remember to think about DNA in this context, but also specifically study...plasmids containing these genes. As with the PIs above, many of these genes were discussed at the 2018 ...chromatin This is not the first Addgene blog post about chromatin--multiple Addgene colleagues and guest...

... dementia-associated tau, delivering dCasRx and a 3-spacer array targeting the MAPT exon 10 splice acceptor...species (read Arbor Biotechnologies' press release about their Cas13d deposit here). Konermann et al.: CasRx...this paper are available at Addgene. Learn more about research at Arbor Biotechnologies. Additional Resources... Resources on the Addgene Blog: Learn about CRISPR RNA Editing with REPAIR Get tips for using Cas9 Transcriptional...Transcriptional Activators Learn about Cas13a (C2c2) Characterization Resources on Addgene.org: Find...

...PMCID: PMC49545. 3. Coffin J, Hughes S, Varmus H. Retroviruses. Cold Spring Harbor Laboratory Press; Cold ...applications to help you start (safely!) thinking about the possibilities. Ready to plan your next (or maybe...Additional Resources on the Addgene Blog Learn More about Lentivirus Listen to Connie Cepko Discuss Gene Therapy...Addgene.org Browse Our Viral Vector Pages Learn About Viral Vector Biosafety Deposit Your Viral Vectors...

...wrong, replace them for free. Plasmids ship out 2-3 days after ordering, making it a toss-up as to whether...cloning before you can start your experiment? What about all the reagents you need to order? Sometimes, it...dive deep into generating the data you’re excited about. When we were closing in on the one million plasmids...million plasmids shared than not!), we think it’s about time we updated this post. How much time does ...choose the right cloning method Find blog posts about scientific sharing here Download the Plasmids 101...

...the insert is large, we may only check the 5’ and 3’ ends to verify that the sequence assembly and identity...One of the best things about sharing plasmids through Addgene is that we provide an added level of confidence...bp of clean sequence from a typical reaction, or about 250 amino acids worth, so make sure the feature ...96-well microplates (identical copies, see image above). We then select forward and reverse primers for...specifically to target this region, as outlined above. Avoid primers that bind within or must read through...

....  For example, the phosphorylation status of SRC-3 defines its association with other members of the ...a broad range of reproductive, developmental, metabolic, and immune response programs. NRs also encompass...gallstone disease, senescent illnesses, and the metabolic syndrome. Coregulators Coregulators constitute... regulatory regime (such as genes involved in metabolism, growth or cytokine action). Primary or core ...and the free exchange of these vectors between laboratories. The development of the transient transfection...