...counting the number of puncta in each cell. When making figures for a paper or presentation, make sure ...binding non-specifically – try optimizing your blocking and washing conditions or consider using a different...concentrations of all the different antibodies, blocking, washing, and sample preparation steps, in order...Immunofluorescence Staining. In W. H. Yong (Ed.), Biobanking (Vol. 1897, pp. 299–311). Springer New York. ...

...scenarios below for a CRISPR experiment which involves making specific knock-out edits in a gene(s) of interest...off-target frequency of 5%. Your experiment involves making a large sgRNA library to thousands of genes and...certain Cas enzymes are less compatible with the bulkiness that accompanies mispairing of bases. To reduce...enzyme swaps will only reduce off targets where nicking/cleaving of DNA occurs, not for deadCas activator...

... single dependent variable. I know what you’re thinking – this really sounds like a two-way ANOVA. And...calculated p values from smallest to largest and ranking them (1 being the smallest value). It calculates... you want to know type of distribution you are working with, graphing data with a histogram is sometimes... friendly neighborhood biostatistician happy by making your statistical analysis plan when you design ...

...eBook collection is a handy resource for anyone looking to use antibody-based applications in their research...: What is an Antibody” quite useful. If you’re looking to design your first flow cytometry experiment,...the Antibody 101 eBook is a great place to start looking for answers.    We hope you find the latest addition...

...Miyawaki, Griesbeck, Heim, & Tsien, 1999), monitoring kinase activity (Ni, Titov, & Zhang, 2006), quantifying...pressure-test your FRET assay to make sure it is working? This article addresses these questions, which ...s best to consider these cases separately when talking about the tools that are available. Let’s consider...Denis V. Titov, and Jin Zhang. "Analyzing protein kinase dynamics in living cells with FRET reporters." ...

...Fly-related Issues in the Lab A significant drawback of working with fruit flies is their ability to escape their...experienced the minor yet consistent annoyance of walking into a fly lab and finding ourselves accosted by...system in a video filmed at Addgene:     If you're working in fly research, I hope you find this new technology...

...viral vector specialist, Rebecca. They will be working with Andrew, the head of our scientific support...Specifically, Jo, Brook, and Angela are co-leading "Making a Greater Impact Using Clear and Accessible Scientific..., Instagram, and Bluesky.   Finally, if you're looking to brush up on your conference skills, check out...

...chemically modify target bases without breaking the DNA backbone, making them efficient and precise tools for... So how have scientists solved this problem? By taking advantage of repair pathways within the cell — ...mammalian cells Researchers in the Xueli Zhang lab were making similar observations around the same time. (The...

...   Unbound Cas9 Now that we know what we’re looking at, let’s peek at unbound—“apo”—Cas9 from Streptococcus... the absence of guide RNA, preventing Cas9 from making off-target cuts.   Pro tip! X-ray crystallography...region between the two (Jiang & Doudna, 2017). By linking the two nuclease domains, Cas9 ensures both are...biology can help you visualize the system you’re working with and better understand how to manipulate it...

...If you’re new to plasmids, we highly recommend checking out the video). Tom has made many more Science...sciencewithtom.com. Many thanks to Tom McFadden for taking the time to sit down and talk with us. Tom McFadden...McFadden is a YouTuber and science educator looking for creative ways to implement the next generation science...

...Thoughts on Making Scientific Information Accessible We'd like to thank Professor Cepko for taking the time...-9:04 : What got Professor Cepko Interested in Working on the Eye 9:05-10:16 : Cell Targeting in Retinal...

...common techniques and concepts. But what if you’re looking for information beyond the 101? That’s where our...our YouTube channel and watch it now! (Bonus: Tim kindly offered to finishing answering all submitted questions...check out our Instagram all summer long to see what kind of shenanigans we get into!  We hope you are as ...

... used to thinking about the downstream results of actions. I’m also pretty adept at thinking about risks...was in the early 80s, and biotech was really just taking off. I found out that patent attorneys with a biology...

...s not the virus itself that kills you, but the cytokine storm it ultimately sets off.  The virus works...only viral protein present on the virion surface – making it critical for viral attachment and fusion. Once...attachment and fusion. Recommended Reading For those looking to delve deeper or stay tuned, there are plenty...

...2018 Microbe meeting. Soon after I arrived, I was looking through a paper-based, affordable, and portable...fascinated by the world that reveals itself when looking down a paper thin and very portable microscope....the scientist in everyone - everywhere! From ‘looking at’ to ‘listening to’ microbes But seeing microbes...

... expand reagent sharing even broader and began working with tech transfer offices to make reagents available...Plasmid Incompatibility by Leah Schiesow If you're thinking about hosting multiple plasmids in one cell, it...this year and along with it, wrote these tips for working with RNA and extracting RNA without a kit. 3. ...

...CRISPR For my editor’s choice picks this month, I’m picking two posts that describe methods to make single ...posts throughout the month including a first of its kind for the Addgene blog - Addgene Software Engineer...Executive Director Joanne Kamens. I highly recommend taking a look at all of our posts as each one has something...

...this interest in the ways science is shared and in making it easier to share, we take special note as the...bump in citations for their trouble, even when all kinds of other factors, including journal impact factor...Way to Share Science, in honor of a decade spent making plasmids more accessible and easier to share. As...

...protein tracking in vivo. Sci Rep. 2015 Jul 7;5:11771. doi: 10.1038/srep11771. PubMed. McKinney SA, Murphy... the 1990s, biologists have spent a lot of time making things glow. Chances are you’ve used a GFP derivative...

...Experiments (JOVE). The video publication by Stuart Orkin and Daniel Bauer's labs details the use of CRISPR...biallelic deletion clones:    Like the video? The Orkin and Bauer lab's related paper describes their methods...Harvard Medical School in the laboratories of Stuart Orkin and Daniel Bauer. He received his BSE in Bioengineering...

...of lab BBQ parties - despite BBQs being wishful thinking in England. The Cepko Lab throws going away parties...celebrate each other's accomplishments? Any tips on making time to relax and/or enjoy time away from the bench... Dr. George Church (Harvard Medical School) for taking the time to speak with us throughout this series...

...write brief descriptions on each. Place the notes sticking up out of the top of the notebooks, so that you...use the search bar to quickly find what you’re looking for. Tip 3: Share protocols and spreadsheets with... knowing the information is backed up and edit tracking will help you identify when others have added ...

...nLightR greatly expand the toolbox for researchers looking to study norepinephrine, the Patriarchi lab continues...Ovchinnikov, S., & Steinegger, M. (2022). ColabFold: Making protein folding accessible to all. Nature Methods...Technique is Best for Me?  In Living Color: The Skinny on In Vivo Imaging Tools Resources on Addgene....

...because it finally feels to me like the postdocs are taking some steps to control their own future in science...speaker, Dr. Henry Bourne (shown in photo), has been speaking out and writing about issues in our science training...experience and encouragement.  Dr. Sibby Anderson-Thompkins, Director of the Office of Postdoctoral Affairs...

...Streptococcus and the “py” in pyogenes), capable of locking two peptide tags together. SpyLigase overcomes ...forces acting on them. For the application we were looking at, magnetic isolation of circulating tumor cells..., highly stable and has this amazing chemistry locking the domain together - no one knows what for. Many...

...Especially when your collaboration includes scientists working in different time zones, it’s important that everyone...accountable to these expectations once they’re set by checking in about them at your recurring meetings and reaching...common. I hope these tips are useful whether you’re looking to improve communication within an existing collaboration...

... cells containing the plasmid construct you’re seeking. Fortunately, there are many ways to do this involving... a successful uptake of your DNA of interest by taking advantage of bacterial characteristics such as ...for specific amino acids. When plated on media lacking the essential amino acid, the bacteria will only...

...also involved in reinforcement learning, decision making, and motor control.  What do we understand about... 100 nm) of DA. In addition, they exhibit fast kinetics (10 ms on and 100 ms off), enabling detection ...magnitudes of fluorescent responses. Studies on the kinetics of these sensors showed rapid response (60 or ...

...fluorescent signal and enables single molecule tracking  within living cells without affecting protein...managed to track run lengths of the motor protein kinesin across microtubules. Tanenbaum et al. then tested...photobleaching Simplified, long-term single-molecule tracking Caveats of SunTag Very large size: a 24x scaffold...

...background noise. This is not terribly problematic when working with cell culture, because it is easy to harvest...harvest large quantities of cells. However, when working on specific tissues or organs in whole animal models...major technical hurdle.   ChIP-seq requires  cross-linking during an initial fixation step. This serves to...

...mapping These tools are for viewing, editing or making plasmid maps, but can also analyze and annotate... Viewer: The free SnapGene Viewer is great for looking at plasmid maps and viewing sequencing traces, ... blank spaces, from the text of a DNA sequence, making it ready to copy and paste into other applications...

...video. Don't worry - no pipettes were harmed in the making of this photo!  Here, I was also given a mixture...such as stuffing new depositor welcome kits, note taking for protocol video production, and keeping inventory... my time here comes to an end, my worries about making connections with coworkers seem, in retrospect,...

...bunch. They don’t really know where I came from.” Working at eScience Labs, an educational company that creates...that provides scientific editing services. “I was working during the day and editing at night. It was not...moved out here, I was still editing from home and looking for a job.” But Caitlin’s persistence paid off:...

...headquarters. I decided to go ahead and apply; thinking I probably would not get selected for both the...compose and submit a manuscript that they were working on currently. Upon arrival to the headquarters,... The most beneficial aspect of this course was breaking up into focus groups with a facilitator. In the...

...expressing a selection cassette with 5′ and 3′ flanking sequences matching the sequences upstream and ...to be assayed (e.g. pPD95_86 and pPD57_56), or knocking down YGOI through RNA-mediated interference (e.g... applications used to study gene function. From making transgenic animals, gene targeting and genome modifications...

... used at high concentrations (and vice versa). Looking around the lab, you'll likely find many of the ...mammalian cells.   Name Class Mode of Action*   Working Concentration** Kanamycin aminoglycoside Binds...their mechanism for killing bacteria, and general working concentrations. For instructions on how to prepare...

... no fear - plasmids from Addgene depositors are making it easier than ever to use CRISPR  in E. coli and...gene body can repress transcription by physically blocking the elongation complex from binding the DNA or...Patino R, Heussler GE, Hearne CC, Qu J, Inclan YF, Hawkins JS, Lu CHS, Silvis MR, Harden MM, Osadnik H, Peters... Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-Guided...

...replication machinery room to come in and get busy making copies.     Types of origins of replication ...compatibility with one or more other plasmids. Generally speaking, plasmids with the same ori are incompatible because...chloramphenicol – make sure your growth conditions aren't working against you!  The culture inoculum Freshly ...

...Addgene started dealing with this new reality of working remotely, scientists around the world mobilized...took to make all this happen with 80% of the team working remotely. We’re extremely proud of the work we’... maintain our workforce for when we need to be working at full capacity to serve the scientific community...

...from Zhu et al., 2019.  Wensheng Wei's lab at Peking University has developed a new CRISPR screening...injection of precise titrations of AAV or heat-shocking the organism. The SPARC system uses competitive...fluorescing green. Image from Shin et al., 2019. Looking for an alternative to intercalating fluorescent...

...around the world who would otherwise have difficulty making or getting these reagents is profound.” Frank Stegmeier...Library, a library that “enables the high-resolution tracking of more than 1 million cancer cells under drug...became passionate about virology,” he says. He was working in an HIV lab and based on that experience, soon...

...species). This feature is advantageous if you’re working with small capacity viral vectors such as AAV. ... “recovering” its original sequence. If you’re looking to disrupt gene function via indel formation, this...targets. Are you using NgAgo? Let us know how it’s working in the comments section! Read About NgAgo User'...

...In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time adding...restriction site (GAATTC) to the 5’ end of this primer, making our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC... get PCR amplification. We can start similarly, taking the final 18 bases of the ORF, including the stop...

...frequently with little notice or choice Always working on a team/in a matrix - can be frustrating for ...but good performers move around easily  Table 1: Taking it a Step Further - Big Company v. Small Company...on what paths to pursue. Spend some time really thinking about your motivations and what parts of being...

...fewer ethical and logistical challenges than does working with animals. Because Arabidopsis is small, has... to grow, it is an ideal organism for teachers looking to expand their science curriculum. Growing Arabidopsis... biology teacher or professor? Whether you are looking to develop a new teaching resource or you want ...

... Why and Where Do I Find One? If you need help making a transition into a new job or maybe to an entirely...gene delivery, but have limited packaging capacity making them difficult to use with CRISPR as the cargo..... 10. Plasmids 101: Control Plasmids Need help thinking about the types of controls you’ll need for your...

...contributed by Eric Perkins Mammalian genomic DNA is not just a 2-D linear molecule snaking through our cells...These loops not only provide a 3-D structure for packing genomic DNA into nuclei, but are also thought to...

...biological motion (whether endocytosis, vesicle trafficking, or muscle contractions) are produced by orchestrated...the ciliate. It’s thought that they function as a kind of armor to protect their host from predation (Petroni...

...Cas9, and the tracrRNA which acts as a scaffold linking the crRNA to Cas9 and facilitates processing of...different components of CRISPR, It's time to start thinking about how you can used them in your experiments...experiments. Use the following flow chart to get you thinking about the types of experiments you're planning on...

...“reading”) of DNA, have changed our collective thinking about the feasible size and scope of projects ...library prep. As part of our mission, we’ve been working with Addgene to develop and apply our plexWell™...read level, and because custom primers or "primer-walking" reactions are completely unnecessary for sequencing...