Retrograde AAV viral preps
Type
Collection
...- pAAV SYN1 HA-hM3D(Gq) Syn Activator DREADD Christopher Richie 50475 pAAV-hSyn-hM4D(Gi)-mCherry Syn Inhibitor...
We narrowed to 728 results for: STO
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CRISPR Pooled gRNA Libraries
TypeCollection...Interest Click on different properties to create a custom filtered list of the CRISPR pooled libraries in... -
Fluorescent Protein Guide: Subcellular Localization
TypeCollection...Harrison 38277 pMXs-puro GFP-p62 Autophagosome Sequestosome-1 EGFP Noboru Mizushima 38272 pMXs-IP GFP-WIPI... -
Mesothelioma - Causes, Symptoms, and Treatment
TypeBlog Post...lung, the heart, and the abdomen. Long story short, when asbestos is breathed in (or less frequently, ingested...small, hook-like shape of asbestos fibers. Over time these embedded asbestos fibers cause inflammation...known safe level of exposure to asbestos – i.e., any exposure to asbestos is potentially capable of causing...exposed to asbestos but have not developed the cancer yet Those who have not been exposed to asbestos Tests...greatly decreased through a ban on asbestos. Nonprofits like the Asbestos Disease Awareness Organization ...bill passes, it will put an immediate stop to the use of asbestos, thus ensuring that future generations...completely preventable. Mainly caused by exposure to asbestos, mesothelioma is a form of cancer that develops... -
Tips for Technical Support Calls
TypeBlog Post...Having spent that long in customer service, I've daydreamed about the ideal customer service interaction -...to any biology-related customer support interaction. Before contacting customer service In an age of Tweeting...answered hundreds of phone calls and thousands of customer service emails in my six years as a senior scientist... - one that gives our customers the most utility in the least amount of time. Though I now spend far less...my years of accumulated wisdom so that you, the customer, can get the most out of your email or phone call... bad product (in which case, you should probably stop using that company). Frequently asked question pages...section are really, truly frequently asked by customers (and I expect this is the case at most companies... -
How Dry Ice Affects Viral Vector Sample pH & How to Avoid It
TypeBlog Post...pH. We compared the dry ice-stored samples to controls that were only stored in a -80°C freezer and not...of tubes: the ones we use to store lentivirus and the ones we use to store AAV. Our results showed that...mitigating the pH effects of dry ice storage. To do this, we stored samples on dry ice for 72 hours and...reading a publication describing how short term storage on dry ice can shift sample pH, we wondered whether...freezer and then moved them to a closed dry ice storage container for 48 hours (to replicate how our viruses...from the dry ice). After 48 hours of CO2/dry ice storage, we then thawed the samples at room temperature...thawed, there was no effect on the pH of the samples stored in our lentivirus tubes (Fig. 1). However, once... -
Tips from the Repository Trenches: Using Barcodes to Track Samples
TypeBlog Post...then inoculated for storage on Thursday. The lab prepares bacterial glycerol stocks on Fridays. From each...used for long term storage (one is kept at Addgene and another is sent to a storage facility). The third...courier and then the journey of transformation and storage begins. Some samples are submitted as bacterial...intact. Some deposits are sent from labs nearby in Boston and Cambridge. Many deposits arrive from labs around...sample can be tracked easily at each step in the storage process. After the original sample is barcoded,...entry into Addgene’s repository. For long-term storage in our facilities, every sample is transferred ... - DNA sent from depositors, bacterial glycerol stocks, prepped DNA ready for our quality control processes... -
Career Insights: Technical Support Specialist
TypeBlog Post...support through the customer’s perspective is good. If you’ve never participated in a customer support conversation...the types of questions customers ask, and there are also differences in customers’ personalities and their...reviewing customer data sets, troubleshooting protocols, analyzing information, and solving customer problems... product issue in the lab. The issue was with a custom-made reagent to manipulate cells (manufactured ...multiple languages - that makes communication with customers from around the world easier. You should also ...expectations. If you are willing to listen to a customer’s concerns and are patient enough to find the real...are the most popular ways to communicate with customers. Online live chat is a convenient alternative ... -
Plasmids 101: Common Lab E. coli Strains
TypeBlog Post...amber (UAG) stop codon by glutamine insertion supF (tyrT) Suppression of the amber (UAG) stop codon by...or protein expression. If you were to include customized strains, the number is probably in the thousands... your plasmid or carrying out your experiment. History of E. coli strains E. coli are gram-negative, rod... DH5alpha and DH10b (also known as TOP10). The history of B strain is a bit more convoluted due to researchers...researchers sharing and renaming samples throughout history. It was likely isolated in 1918 but was first referred...Streptomycin MC1061 derivative. General cloning and storage, blue/white screening, leucine auxotroph. F- endA1...mrr-hsdRMS-mcrBC) λ- DH5alpha General cloning and storage of common plasmids, blue/white screening. F- endA1... -
The Challenges of Cell Culture
TypeBlog Post...grow this initial stock and create a set of vials of ‘Master’ stock, which can be stored in liquid nitrogen...some of these vials will be stored in a separate backup liquid nitrogen store to prevent loss from equipment...used to produce a set of ‘working’ stocks of cells. Working stocks should be frozen in multiple vials ...plenty of maintained freezer stocks. It is vital that the Master and Working stocks of cell lines have the ... monitor the stock levels you have in liquid nitrogen for your Working and Master stocks of each cell ...and money but cells lose provenance (this is the history of the cell line consisting of records of the origin...used up. This ensures you can retain the maximum stocks and use of the original Cell line you purchased... -
Flow Cytometry Readouts: Yes, No, and Everything in Between
TypeBlog Post...dot plot, looking at a histogram vs antibody plot would be better. The histogram shows peaks of high signal...gates, just based on a histogram plot instead of a dot plot. However, the histogram plots look a bit different...lower signal, or downregulation. Figure 3 shows a histogram for a yes-no dot plot, like the one shown in Figure... Figure 1. Figure 3: An example of a histogram plot for a yes/no flow cytometry readout. Created...Created with biorender.com. Using histogram plots to quantify smeary plots For smeary expressions, ...for a signal peak that shifts to the right on a histogram plot and lower for a signal peak that shifts to...for yes-no readouts. Figure 4: A histogram plot for a smeary antibody readout. The orange... -
10 Steps to a Perfect Science Talk
TypeBlog Post...point. What makes a story? A story is a narrative connecting cause and effect. Stories can and should be... or notes. Listen to their stories and narratives (see below “Tell a story”) and note how they build suspense...hear material delivered in the form of a story. Using stories is one of the best ways to make you and ... a lab meeting (maybe not there either). Think Keystone Keynote presentation or Ted Talk. You want to ...me and wished I had been more careful. 3. Tell a story We are biologically programmed to listen and remember...presentations engaging and memorable. If you read my story above about the presentation faux pas, you are more... striking images, creative ideas, and engaging stories as you encounter or experience them. Drop content... -
Illuminating Epigenetics with A FRET Based Biosensor
TypeBlog Post...substrate (the histone-derived peptide) which can localise the construct specifically to the histone protein ...target the K9 (pcDNA3-K9 histone methylation reporter) and K27 (pcDNA3-K27 histone methylation reporter) ... remodeling. Chromatin is a complex of DNA and histone proteins that compacts cellular DNA into chromosomes...dangerous DNA damage. If the way DNA is wrapped around histones changes, then gene expression can change as well...The more frequently and tightly bound DNA is to histones, the more compacted it becomes. This prevents ...can occur by two main mechanisms, either through histone modifications, which can influence the transcription...plasmid-based biosensor to visualise changes in histone methylation in living cells. Essentially this reporter... -
Twenty Years of Addgene Sharing: CRISPR
TypeBlog Post...clear that it’s also a story of community, cooperation, and sharing. Sharing this story is a truly special...celebrate twenty years at Addgene, we are sharing stories of our repository’s positive impact on science ...science and scientists around the globe. Our first story focuses on a technology that has changed virtually ...libraries, and they remain the most popular with our requestors. However, we can again see quick adaptation and...better. We therefore looked at depositors and requestors by country, and graphed the top ten countries...surprise that the USA is the largest depositor and requestor of CRISPR plasmids. But many other countries, ... to Australia, are significant depositors and requestors. CRISPR tools are developed and used all over... -
An Inside Look into Addgene's Dev Team
TypeBlog Post...into small, well defined chunks, called stories, and work on stories during short periods of time called ...clarifications lead to the story being scrapped or broken up into smaller stories. Since this occurs before... sprints. The team is mindful of the number of stories we commit to for each sprint, to ensure we will...process for working on a new feature. We start with a story that describes the feature and what the criteria...accepting the feature as done correctly. These initial stories are usually a result of collaborations between ...stakeholders (other Addgenies). Once we begin work on a story, we continue to collaborate closely with each other...the new feature as done and we’re onto the next story. Figure 1: Addgene’s Agile Approach Sharing... -
How to Prepare for an Industry Interview
TypeBlog Post...for drafting my stories. In total, I prepare 5-6 stories for an interview, since one story usually has more...Instead, I preferred to prepare stories to answer interview questions. Story telling shares many of the same...of the theme of my interview stories as the characteristic which the story demonstrates I possess. Bullet...helpful for my interviews in industry. Prepare stories to tell in your interview The STAR interview method...answer: a setting, a conflict, and a resolution. Stories usually have a theme too, which is something not...of questions. Writing a rough outline of these stories helped me remember them for the interview. Prepare...problem, or making a key decision. Even deciding to stop pursuing a particular line of research is a deliverable... -
5 Tips for Troubleshooting Viral Transductions
TypeBlog Post... researchers prefer to store freshly harvested virus at 4 °C instead of storing it briefly at -80 °C. ... in a given viral stock will give you a sense of the infectivity of your viral stock, save you time, and... compare it to some reliable controls. 2. Viral stocks may be sensitive to freeze thaw cycles. Depending...these can be increased by concentrating a viral stock. Different titering methods can reveal differences... your virus by ultracentrifugation of the viral stock, followed by resuspension of the collected particles...highly sensitive to freeze-thaw cycles and should be stored in single-use aliquots. Never use a polybrene solution...increase transduction efficiency by 1.5-fold (Stockschläder et al., 1999). 5. Check your packaging cells... -
10 Basic tips for mammalian cell culture
TypeBlog Post... them, and store all but one in liquid nitrogen. The frozen vials make up your master stock, while you...be used as your working stock. This way you can go through your working stock vial by vial before thawing...another vial of the master stock. This ensures that you have many cells stored in their original condition...Keep the hood clean Hoods should not be used as storage cabinets. Clutter not only makes it difficult to... make things more manageable, but it also keeps stock solutions from getting contaminated. Make sure cells...like their space. If they reach confluence, they stop growing (contact inhibition) and it takes them time...condition and can replace your working stock if it gets too old or contaminated. This also ensures that anyone... -
Addgene's Tips for Plasmid Quality Control
TypeBlog Post...glycerol stock production and storage, we grow a little extra and transfer 100 µl of glycerol stock to each...that we are sequencing from the exact same stock that we store and distribute from. Of course, your culture...software such as Primer3 to help you design your custom primers. Remember, when designing a sequencing ... of our Sanger sequencing is done from glycerol stocks (overnight cultures with 15% glycerol), as opposed... culture. Sequencing directly from the glycerol stock saves us the step of purifying the DNA and gives...96-well PCR plates are prepared with the primer stocks for forward and reverse reactions, respectively...equipped to perform Sanger sequencing from glycerol stocks, so make sure to select the appropriate service... -
CRISPR 101: Epigenetics and Editing the Epigenome
TypeBlog Post...field in which covalent modifications to DNA or histones, the proteins that help package DNA, were associated...modifications, researchers used blunt tools like histone deacetylases, but targeted epigenetic modification...researchers additionally fused a TAL effector to LSD1 histone demethylase to demethylate enhancer regions (Mendenhall...regulate transcription, also includes light-regulated histone methyltransferases and deacetylases. CRISPR and... acetyltransferase increases levels of H3K27ac histone modification at specified loci. Charles Gersbach... Figure 1: dCas9-p300 adds H3K27ac marks to histones in promoter and enhancer regions. These marks ...and repair pathways work downstream of Tet1 to restore the DNA after cytosine removal. Recently, Albert... -
CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics
TypeBlog Post...bound to modified proteins called histones. We can detect modified histones with “GPS for proteins,” ChIP-... of modified histones, a genomic region that has mono-methylated and acetylated histones, but not tri-...map with three coordinates: chromosome, start, and stop. For instance (chr3, 1, 10) indicates a stretch ...Locates ChIP-seq Proteins (for our example later, histones) ChiRP-seq RNA Hi-C DNA (genome-to-genome ...tri-methylated histones, functions as an enhancer. We can therefore denote enhancers as follows: (mono_methylation...of epigenomics: H3 – one of NANOG’s associated histone proteins K4 and K27 – locations of the amino ...usually housed in a single flask. Through a bit of stochastic magic and Poisson statistics, each sub-population... -
27 Hot Plasmids from 2016
TypeBlog Post... are provided in both open (no stop codon) and closed (including a stop codon) formats. The collection..., et al. Cell Rep. 2015. PubMed PMID: 26190106 PRESTO-TANGO: An open-source resource for drug discovery...receptors. Bryan Roth's lab has a newly available PRESTO-TANGO kit which allows researchers to interrogate...and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library of approved ...discovery. Researchers can use plasmids from the PRESTO-TANGO kit directly for drug screening or easily...Luckily, researchers from Douglas Densmore’s lab at Boston University have greatly simplified the process ...CIDAR MoClo Parts Kit are available as glycerol stocks in a single plate with every plasmid or as individual... -
Getting the Most from Your Lentiviral Transduction
TypeBlog Post...thawing a fresh vial. In many labs, the history of the cells in storage may be a bit dubious due to lack of... especially when the lentiviral stock needs to be titrated. If storing for less than a day, lentivirus...For long-term storage, viral preps should be divided into single-use aliquots, and stored at -80°C. Some...want to consider concentrating your viral prep. Storage can also play a role in the success of your lentiviral...factors such as the transgene expressed, the viral storage buffers used, and other experimental parameters...dry ice/ethanol bath or liquid nitrogen prior to storing. Know your target cells Labs tend to follow the...serve as the cellular receptors for vesicular stomatitis virus.” Proceedings of the National Academy of... -
Data Freedom: The Expansion of Data Sharing in Research Publications
TypeBlog Post...publishers. Nonetheless, the initial release caused a storm as the organization left open the question of how...for advice about the scope of the policy. End of story? Not quite. The PLOS policy on data access is a...statistical methods, omission of some data, etc. The firestorm that followed implementation of the policy comprised...easily available, organized in a form that can be understood by others, balanced against how often such data...of research, raw data requires huge amounts of storage. For example, mouse behavioral geneticists may ...data should be included? We should recognize that storing data in a sharable form is indeed a significant...carried out. While no one is suggesting all data be stored in a retrievable format, we too rarely preserve... -
Why and how to start studying chromatin (with Addgene's help)
TypeBlog Post...were discussed at the 2018 Keystone meeting for Chromatin. Note that histone genes frequently have many...context of chromatin?” Though it’s true that adding histones and chromatin architecture to every diagram portraying...Chromosome Architecture and Chromosome Organization Keystone Symposium. As it happens, there are a lot of scientists...transcription of a gene a megabase away, or looking at the histone methylation status at a double-strand break that...the presentations and posters I saw at the 2018 Keystone Chromosome Architecture and Chromosome Organization...Sharon Dent Archa Fox Elaine Fuchs Kai Ge Robert Kingston Thoru Pederson Phil Sharp Julie Ahringer Alexander...RANbodies) as versatile, small, sensitive immunohistochemical reagents. Yamagata M, Sanes JR Proc Natl ... -
Supporting Reproducibility with a Connected ELN
TypeBlog Post..., Word docs, and the like is likely stored in online file storage tools like Dropbox, Box, One Drive, ...commonly used online file storage tools, as illustrated in this video, and to data stored on institutional and...like image banks and sequencing files is likely stored in institutional and lab servers specifically designed... conveniently reference specific pieces of data stored externally in the record of research compiled in...fully supported API that enables development of custom integrations with other tools and resources. As... in the documents to data in the external file storage resources remain live. This means that someone ...research and commercialization community in greater Boston, while at the same time keeping one foot on the... -
Tips and Tricks for Using Golden Gate Modular Cloning (MoClo)
TypeBlog Post...CDS1, coding sequence with stop codon; CDSns, coding sequence with no stop codon; TAG, C-terminal tag;... own glycerol stocks for more frequent use. This way, if one of your new glycerol stocks gets contaminated...in your laboratory as plates of frozen glycerol stocks. It is very important that these stay frozen at...rounds of freezing and thawing will affect bacterial stock viability. When handling your MoClo plate, always...preferably on dry ice. Careful use of your glycerol stocks When your lab adopts the MoClo technology, you ...you will quickly go through your plasmid stocks. To avoid cross-contamination, we recommend retrieving plasmids... miniprep a lot of plasmids from your bacterial stocks beforehand. You can also use high-throughput methods... -
Antibodies 101: Avoiding the Mouse on Mouse Mess in IHC
TypeBlog Post...human cryostat tissue sections. J Histochem Cytochem : Off J Histochem Soc 38:923–926. https://doi.org/...Fighting with antibodies to produce immunohistochemistry images that are crisp, bright, and lacking in...avoiding background secondary binding Direct immunohistochemistry (Figure 2A) How do you avoid having your...Randolph-Habecker J (2013). A Flexible Mouse-On-Mouse Immunohistochemical Staining Technique Adaptable to Biotin-Free...Immunofluorescence, and Multiple Antibody Staining. J Histochem Cytochem 62:197–204. https://doi.org/10.1369/0022155413511620...Monoclonal Antibody to Mouse Tissue Sections. J Histochem Cytochem 46:977–983. https://doi.org/10.1177/002215549804600813...Tuson JR, Pascoe EW, Jacob DA (1990) A novel immunohistochemical technique for demonstration of specific binding... -
A Quick Guide to a Career in Software Product Management
TypeBlog Post... it out. 4:00 PM--Respond to customer help tickets. Catch up on customer help tickets, and respond when...advocate for the customer and help build software and technology tools with the customer in mind. You can...Oxford Nanopore Technologies, I work with our customers in the US to learn where the gaps are in their... so many different people! I communicate with customers, software developers, as well as team members ... It’s divided into the key takeaway themes and customer pain points we’ve identified. I can’t wait to ...know what to work on next to help alleviate any customer pain points. Many thanks to our guest blogger... -
Celebrating 15 Years of Scientific Sharing
TypeBlog Post...usual quality and success. Customer and technical support Our customer and technical service brands...helpful and the customer and technical service teams put 100% effort into helping our customers every day in...have for the future. At Addgene we will never ever stop planning, perfecting, and learning, but there are...scientific fields.” Thanks to our legal team, and great customer service, somehow we have scientists actually loving...offer online for Addgenies and to serve external customers. I also can’t wait to see where our new data initiatives...to do it quickly. Hiring is hard but as a 3 year Boston Globe and BBJ Best Place to Work with a positive...