...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...

...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...

...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 ...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

... 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...

... 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...

...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...

...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...

...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...

...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...

...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...

... 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...

...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 ...