... by ultracentrifugation (75,000 - 225,000 g for 1.5 – 4 hours at 4 °C). After ultracentrifugation, virus...integrity of your virus, perform a diagnostic restriction enzyme digest and compare it to some reliable...vectors) you can concentrate your virus by ultracentrifugation of the viral stock, followed by resuspension...packaging cell debris before proceeding to ultracentrifugation to avoid contaminating your transduction ...through a 0.45 µm or 0.22 µm filter) or by a centrifugation step (5 minutes at 300-500 g). After removing... to concentrate a virus solution without ultracentrifugation, you can reduce the volume of cell culture...

...days Purification Ultracentrifugation (AAV); centrifugation (lenti) Centrifugation Titration time ~...using methods like ultracentrifugation, while lentivirus supernatants are centrifuged and filtered to eliminate...have a higher titer than a P1 virus. Pro-tip! Centrifuge the viral supernatant in between passages to ...straightforward — simply spin down the supernatant in a centrifuge to remove any unwanted cellular debris, as you...

...and sensing electrical potential. Thankfully, there is a tool to measure these electrical events! Voltage...biological tools. Why Use Voltage Indicators? Electrical signals are integral to many facets of biology...muscle contraction – is directly related to the electrical impulse of the cardiac cells. To study this phenomenon...is carried out by action potentials which are electrical charges propagated through multiple cells to ...depends on the ability to monitor their signals – electricity! The research tools for studying voltage within...was dipicrylamine, a lipophilic compound that distributes itself within the plasma membrane as well. Another...

...This post was contributed by Jake Watson and Javier García-Nafría from the MRC Laboratory of Molecular...present even in common lab cloning strains. This intrinsic bacterial recombination pathway, referred to as... longer oligos, which could be avoided using restriction enzyme cloning, were more prohibitive at the ... doesn’t matter if the DNA is PCR amplified, restriction digested or even a synthesized gene. PCR is the... Mutagenesis is performed by designing primers astride the old codon, amplifying the whole vector. The...extending homologous sequences (to 25-30 bp). Tips and tricks for IVA cloning Use a sequence manager A sequence...IVA can be employed by cutting the vector with restriction enzymes and adding the corresponding homologous...

...colonies in petri dishes, but close to 80% of samples are received as DNA in a microcentrifuge tube. Once...sample enters Addgene’s lab, a series of events is triggered. Each step is tracked through barcoded tubes that...track all of our 40,000 samples available for distribution!    Check Out How We Think about Developing...The preferred E. coli strains for storage and distribution at Addgene are DH5α, NEB stable, ccdb survival...Each sample that is transformed is plated onto a petri dish plate that is also barcoded. The barcodes on... to be transformed with a micropulser using an electric charge to help the DNA enter the cells. Some samples...

...This post was contributed by guest blogger Derek Simon. The actual experiments you do will be determined...implanting an electrode and non-specifically electrically stimulating an ill-defined region of the brain...trained to “freeze” upon anticipation of a mild electric foot shock that has become associated with a visual...using HR was found to block the acquisition and retrieval of contextual fear memories [5].  In a modified...other regions of the brain may be involved in retrieval of remote contextual memories. This study is an...real-time inhibition), and behavioral (remote memory retrieval) specificity. Optogenetics can be used to more...2012): 159-172.5. Goshen I, et al. Dynamics of retrieval strategies for remote memories. Cell. 2011;147...

...antibody (N206A/8 TC Supernatant). Image attribution: James Trimmer, UC Davis.   ...interact with the air-water interface (AWI) before vitrification, they can be completely denaturated (left) or...representative micrograph (C), particle orientation distribution and Fourier-shell correlation curve (D), and...protein. While the addition of LEAs may bias the distribution of particle orientations, this could be overcome...off-target editing. Cargo size limits are also less stringent for eVLPs than other viral delivery methods, allowing...anti-GFAP [N206A/8R] antibody shared by James Trimmer’s lab (Figure 5) as well as antibodies for identifying...

...study a matrix metalloproteinase: López-Pelegrín, Mar, et al. "A novel mechanism of latency in matrix metalloproteinases...Addgene. What you might not know is that Addgene distributes curated collections of plasmids as kits with ...This kit is designed to be used with Type IIS restriction enzymes and standardized primers to clone your... pExpression vectors) plasmids. The type IIS restriction sites used in these vectors are of low abundance...Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium...used to generate these fusions all use the same restriction enzymes for cloning. Furthermore, some plasmids...

...collaboration. Together, we distributed 184,824 materials to 39,529 scientists across 86 countries. Even more remarkably...remarkably, 1,160 labs from 522 organizations in 41 countries enriched our repository with 15,020 new plasmids...part of the story. This year, we made significant strides in making science more accessible and reliable....thoroughly characterized antibodies, and our contributions to key research papers are already inspiring...Errington, T. M., Turn, R. E., Bandrowski, A., Trimmer, J. S., Rego, M., Freedman, L. P., Ferrara, F.,...

...of months. Read on to discover what new post contributed the most to this record breaking month and to... Apps for Lab Life This very useful post was contributed by guest blogger Sean Stacey from Eastern Tennessee...Phage Genomes for Host Binding Proteins," was contributed by guest blogger Jessica Sacher from the University...Disease, Running for FOP, Running for AJ,” was contributed by Kurt Swanson, husband of Addgene Director ...currently on my way back from a very successful Outreach Trip in San Francisco and would like to give my thanks...who spoke with me and deposited plasmids on this trip. Here’s to hoping that October is even more awesome...

... and were only available as green probes. The Patriarchi Lab aimed to overcome these limitations by developing...vivo experiments, and nLightG was successfully trialed in two-photon in vivo experiments. nLight indicators...nLightR, both generating a measurable signal after electrical stimulation with a single pulse, while nLightR...researchers looking to study norepinephrine, the Patriarchi lab continues to work on further improvements...nLightG. Many thanks to Mike Waring and Tommaso Patriarchi for their assistance with this blog post. References...Benke, D., Weber, B., Bohacek, J., Reiner, A., … Patriarchi, T. (2023). Sensitive multicolor indicators for...

...includes 1049 community-validated reagents from 20 contributors, including 61 fluorescent probes, representing...for the performance of a reagent, e.g., antigen retrieval conditions, dye inactivation details, tissue preservation...   Figure 2: Screenshot showing reagent entries with associated metadata adapted from the Affinity...preservation, target tissue, detergent, antigen retrieval conditions, and more. Users can also recommend... showing the IBEX Knowledge-Base, with every contributor listed as an author.  The IBEX Knowledge-...and share their validation work. Everyone who contributes to the IBEX Knowledge-Base is named on the Zenodo...

... In 2023, Addgene distributed 188,729 materials to 39,844 scientists in 88 countries around the world....1,106 labs in 504 organizations from 37 different countries, into our repository, while expanding global access... access to our repository through our distributor partners, with the launch of antibodies in China and...Protein Innovation (IPI), in which Addgene will distribute recombinant antibodies (rAbs) developed at IPI... repository, partially through work with our distributors in China, Korea, Japan, India, and Brazil. Through...

... Kolmer-Agduhr cells was sufficient to trigger the symmetrical tail movement unique to spontaneous slow...periodically beat their tails left and right in a symmetrical motion to move forward. This behavior is not ...behavior studies in action: Identifying neurons that trigger spontaneous swimming  Researchers at the Isacoff... zebrafish larvae to identify new neurons that trigger the CPG (central pattern generator) (Wyart et al... photostimulation in order to drive the full symmetrical response.  The function of Kolmer-Agduhr cells...

...sequencing results match sequence information contributed by the depositor, published backbone sequences...additional verification experiments: Primer and restriction digest design Some regions of plasmids are difficult...present in the plasmid, we might need to perform a restriction digest to confirm the size of the plasmid and...including our most popular primers and certain restriction enzymes, but you will also be able to create ...annotations and design additional experiments like restriction digests. We’ll have a tutorial showing all of...Basic Molecular Biology Protocols Diagnostic Restriction Digest Protocol ...

...the list weekly. The Blue Flame Award, first distributed this year, serves to congratulate researchers...one plasmid deposited at Addgene that has been distributed more than 100 times. We hope these desktop trophies... or personnel to constantly prepare them for distribution. One of the major advantages to depositing materials...Laboratory in Norwich, UK: “We’re very pleased to distribute our plasmids via Addgene. Given the increasing...world. “It’s easy to imagine how hard it is to distribute thousands of plasmids across the world from the...has made it possible to eliminate geographical restrictions in life science research." Claim Your Award ...

...plant hormones strigolactones By Fabienne Furt Current methods used to detect strigolactones (SLs) are costly...build and validate a ratiometric fluorescent biosensor based on the parasitic Striga hermonthica SL receptor...editing in organoid Biosensors for detecting strigolactones The CRISPR Corner New ready-to-use viral preps... catalytic activity.  As SLs are also known to trigger seed germination of parasitic plants, this approach...

... submissions via email: [email protected] Entries include accession number, species, and assigned...cell lines profiled in the lab. You can browse entries by cell line groups, such as their vaccine production...collections.  Depending on available information, entries may also include whether a cell line is known to...programatically, their API allows fine grained query and retrieval of all the information in the resource.  This ...cells. Image courtesy of Josef Reischig.  Contributing to Cellosaurus Generated a new cell? Or finding...sent the updated information to the cell line distributor. A success all around! If you’re excited about...

...transport at the cell surface Article contributed by Marcy Patrick  Listen to the nanobody toolkit podcast...highly processive reverse transcriptase Article contributed by Will Arnold  Listen to the MarathonRT podcast...occupying a specific genomic locus: Caspex  Article contributed by Susanna Bachle  Listen to the Caspex podcast...occupying a specific genomic locus: C-BERST Article contributed by Klaus Wanisch  Listen to the C-BERST podcast...dCas9–APEX2 biotinylation at genomic elements by restricted spatial tagging), to confirm the composition ...

... This post was contributed by guest blogger Courtney Price, the Education & Outreach Specialist for the...mission is to collect, preserve, reproduce and distribute seeds, DNA and other resources for Arabidopsis...100,000 samples to researchers and educators in 60 countries each year. Introducing Arabidopsis Arabidopsis...fluorescent lights. Other experiments require only seeds, petri dishes, and filter paper. Another benefit of using...learning. Whether visiting classrooms, hosting field trips, conducting teacher workshops, or staffing a booth...partnerships have grown to include local school districts, professional education associations, and local...