...purposes of labor laws, rather than as trainees in a special category, as has been the ‘labor-of-love’ philosophy...passing and upcoming implementation of the Federal Labor Standards Act (FLSA) which mandates that all workers...event of note is a ruling in August by the National Labor Relations Board to allow graduate students who work...

...general consideration for behavioral testing in a laboratory, a high level of experimental control is required... after stereotaxic injection. Find more details about choosing AAV or lentiviral vectors in our previous...it’s crucial to appropriately time the injection about two weeks before the behavior test. This time window... Viral Vectors Resources on Addgene.org Learn about Addgene's viral service Find viral protocols and...

...blog Learn about the advantages and disadvantages of using monoclonal antibodies. Read about other techniques... require two different antibodies. As mentioned above, it can also be used to characterize antibodies ...concentration of proteins in an ELISA well As mentioned above, the measurement of the concentration of proteins...

... are nice if you can afford them). I am talking about making sure the audience is interested, educated...of the stories or topics you know they can speak about. There should be some spontaneity, but having a ...location and parking information and remind them about the event as it approaches. Room set-up and logistics... make or break the discussion section. And what about those audience members that want to take over? It...

...specific genes. Many years ago, people in the laboratory became frustrated with using glass cloning rings...the hood. A 48-well plate is also prepared with about 200 ul of culture medium containing serum in each...incubator, additional growth medium is added to about halfway up each well. Tips for using the agarose...with the firmer 2% agarose gel solution discussed above for picking. Many thanks to our guest blogger Iris...

...the context of the cell. However, much knowledge about functionality can be gained by understanding the...subcellular marker proteins, it’s possible to get an idea about the subcellular structures your protein resides ...with. These “colocalization” studies give insights about the proximity of two proteins within the same subcellular...Additional Resources on the Addgene Blog Learn More about Plasmids for Endogenous Gene Tagging in Human Cells...

...technology. To dig more into what researchers felt about NgAgo, I conducted a survey and asked researchers...successful, and the second section asked basic questions about the NgAgo protocols used by respondents (transfection...27136078. 2. Burgess, Shawn., et al. "Questions about NgAgo." Proteins & Cell (2016). 3. "NgAgo: Genome-editing...PMC4343198. Resources on the Addgene Blog Learn More about NgAgo Enhance CRISPR Targeting Specificity with ...

...of the hood while work is in progress.Learn more about cell culture contamination in this post from the... bath constantly at 37°C for this purpose.Learn about the effects of CO2 on sample pH Cell morphology:... Set up a routine to split cells Don’t be lazy about splitting cells, instead try to form a routine. ...secretion of lactic acid (a by-product of cellular metabolism that is toxic to the cells), and need to be split...

...If you have ever worked in a molecular biology laboratory you have likely done a polymerase chain reaction...uses ddPCR for AAV titrating. You can learn all about ddPCR for AAV titration and find helpful tips and...method that can help save time and effort in the laboratory.  There are two main categories of multiplex ...use a single pair of primers, like those stated above. Multiplex PCR is also used in high throughput SNP...

...directly back to your specific goal. When thinking about step 3, however, you may be surprised at the number...Delivery methods for mammalian cell lines As mentioned above, the expression system you choose in many ways dictates.... The user should review the current literature about their preferred model.  If your expression system... Check out our CRISPR Featured Topic Page Learn about CRISPR Delivery Using Ribonucleoproteins (RNPs) ...

...genes above with your gene or genes of interest. Some of the plasmids listed in the table above (and their... an internal region of the mRNA. In the figure above, you can see that the IRES element acts as another... first discovered in picornaviruses, are short (about 20 amino acids) and produce equimolar levels of ...

...Read this blog post about CRISPR Kinome Libraries available at Addgene Learn about the advantages of pooled...large-scale functional screens Read this blog post about genome-wide screening using CRISPR Resources on...libraries for the mouse genome! Other Resources Read about CRISPRi and CRISPRa from the NIH E-newsletter ...

...gene therapy applications. Read on to learn more about the current (and future) applications of lentiviral...do both, Addgene depositor Didier Trono and collaborators turned to the lentivirus HIV-1, well-known to...Libraries Enable Genome-Scale, Knockout Screening Read about Genome-Wide Screening Using CRISPR/Cas9 Additional...Addgene.org Check out Our Lentivirus Guide Learn about Virus Biosafety Find Lentiviral Vectors for Your...

...CRISPR Featured Topic Page Learn about Non-Homologous End Joining Learn about Base Editing Read 3 Tips to ... post on increasing HDR efficiency to read more about optimizing this distance. Homology arms and types...screening strategies for your experiment. Also learn about Addgene’s knock-in plasmids for reagents to help...

...genome editing than Cas9 - read on to learn more about Cpf1 and check out our CRISPR guide for a refresher...mediated cleavage are 42-44 nucleotides in length, about the same size as Cas9’s, but with the direct repeat... Cpf1 was first identified, we didn't know much about its on-target and off-target editing efficiency....CRISPR system proved we had a lot more to learn about CRISPR biology. Later work, like the adaptation ...

...on the Society for Laboratory Automation and Screening (SLAS) Electronic Laboratory Neighborhood.   Work...worry less about how they will handle an emergency and this will make you much more confident about their ...

...all, a delivery method. I would be remiss to talk about them without looking at what they’re delivering .... But in the years since, we’ve learned so much about what it takes to make these viral vectors not only...., 2016), video, educational resources, and collaborations, and will continue to do so, in the hopes that...leave a comment; or take our AAV survey. Learn more about GCaMPs and other tools developed through the GENIE...

...Resources on the Addgene Blog  Read more about Cpf1 Read more about SaCas9 and AAV Check out our CRISPR Featured... and VRER) recognize novel PAM sequences (shown above). The VQR, EQR, and VRER Cas9 variants are capable...example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged...

...Addgene Blog Learn about using toehold switches to detect Zika virus   Learn more about targeting RNA with...time-consuming, and complex to use outside of standard laboratories. In the case of the COVID-19 pandemic, qPCR ...with Cas13a (C2c2) Learn more about Cas12a (Cpf1) and its multiplex genome editing abilities Resources...

...RSpace is integrated with most of the tools noted above, and has a modern API. Integrations with additional...into RSpace. The kind of informal conversations about research that can take place in Slack normally never...RSpace documents to external data sources (described above) further enhances the value the repository integrations...Minisymposium on Reproducibility Read all blog posts about scientific sharing Resources on Addgene.org Find...

...that lead to these preferences and what can we do about them? Read on to find out!   Why do organisms have...understood, but some possible reasons include: Metabolic pressures - it takes cellular resources to produce...amino acid I’d like to encode,” but, as described above, not every region of a protein should necessarily...Effects on transcription (remember, it’s not all about translation - e.g. codon choice may interrupt transcription...

...resistant infection or not, because scientists in the laboratory will need to isolate and grow whatever bacteria...method that is frequently used in the clinical laboratory. What can we do to stop the emergence of antibiotic...CDC. Antibiotic resistance can be scary to think about, but we can all do our part to learn all we can,...drug resistance. Additionally, she is passionate about health equity, science communication, and public...

... organisms (Chavez et al., 2016). To learn more about the different types of Cas9 activators, check out...covering VP64, Suntag, SAM, and VPR. Worry less about off targets Unlike Cas9 cutting activity, off target...of the Addgene Scientific Advisory Board. His laboratory at Columbia University is focused advancing scientific... Variant for Your Next Experiment Find out More about Genome-wide Screens with CRISPR/Cas9 Learn How CRISPR...

... an AAV rookie  Learn about some considerations when using AAVs Learn more about using AAV as a tool for...PubMed PMID: 29175632. If you want to learn more about how PHP.B and PHP.eB were engineered, check out ...Additional resources on the Addgene blog Learn more about PHP.eB, and PHP.S, a peripheral nervous system (...

... to target individual genes can be relatively laborious because multiple plasmids/viruses are needed to...screening strategies Although the tools described above make CRISPRa more accessible to neuroscientists,...vitro and in vivo applications, the tools described above make CRISPRa in the brain relatively easy and accessible... CNS using CRISPRa as a member of the Gelbard Laboratory at URMC.     References Liu, Yanxia, et al. "...

...uses of biotinylation in molecular biology! Learn about in vivo biotinylation of bacterial fusion proteins...covalently attached to the active site of key metabolic enzymes including biotin carboxylase and decarboxylases... organic acids to produce important cellular metabolites. Biotin acts as a carboxyl carrier from one compound...such as gluconeogenesis, lipogenesis, amino acid metabolism and energy transduction. Biotinylation is the...

...mutations When thinking further about the distance relationship described above, we realized that, in addition...efficiency already drops by half and after only about 30 bp, it is no longer feasible to incorporate mutations...Additional Resources on the Addgene Blog Learn about High Fidelity Cas9 Variants How to Design Your gRNA...

... all desired codon changes (step 1 in the image above). A second universal primer is used to synthesize...first followed by the top strand. Both produced about the same number of positive clones. See Supplementary... of DNA degraded in the first digest (step 1 in above diagram). Remember that the BbvCI enzymes nick the...mutagenesis with the same protocol. What do you think about one pot saturation mutagenesis? How would you use...

... one of the MEK1 phosphomimetic residues in the above study with phosphoserine (to create a pSer/phosphomimetic...non-phospho species on PhosTag gels as described above. Reagents and protocols Sep-OTSλ plasmid - Addgene...interested in the intersection of cell signaling, metabolism, and the mighty mitochondrion. You can follow...Blog Verify Your Constructs with Colony PCR Read about Tags for Protein Purification Resources on Addgene.org...

...deleting residues. Only about 10 amino acids can be deleted from the C-terminus and about 5 from the N-terminus...cylinder with a length of 4.2 nm and a diameter of about 2.4 nm (Hink et al., 2000). The complete beta-barrel...based on cofactor binding proteins as described above. The smallest tags are peptides that are engineered...

...lymphocytes in a mouse tumor sample You made it! So much about the theoretical background. Let’s dive into the ...often choose a looser gate here. I am not worried about including irrelevant events in my analysis, as these...population, acquired from the previous gating pipeline, about 53% of my cells are dendritic cells and ~13% are...ve covered in this blog post. First, you learned about the types of scattering that occur when a cell is...

...roles of individual integrins. The Ha lab — in collaboration with the Springer lab, the Jaumouillé lab, the...recombinant antibodies and plasmids. To encourage collaboration and education in protein science, IPI hosts ...events and training courses on technologies and laboratory techniques related to antibody discovery. You...the Institute for Protein Innovation. She writes about all things IPI, from the scientists and scientific...

...screen (Koveal et al. 2022). Lactate is a key metabolite in glycolysis, so this sensor will be a valuable...valuable tool to study cellular physiology and metabolism under various conditions. LiLac has improved response...going viral! In addition to the viral preps noted above for iGluSnFR3 and JEDI-2P, several biosensors already...innovation next year! Did we miss any? Let us know about your favorite new biosensor plasmids in the comments...

...compile more comprehensive demographic information about the postdoc work force, problems start with the ...by Paula Stephan is $40,000) Increase Knowledge about Postdoc and Graduate Student Career Outcomes University...through many iterations read by your PI, your collaborators, others you know in the field, and even people...important questions that still need to be answered about the makeup and welfare of the many researchers out...

...integration of multiple copies of the construct (about 8 in Brainbow-1.0 mice.) The combinatorial effect...with the goal of overcoming the limitations listed above. First, they screened a variety of fluorescent proteins...method has become very useful in lineage tracing; “Zebrabow” was used to trace the development of the corneal...: 10.1038/nmeth.2450. Epub 2013 May 5. PubMed. Zebrabow: multispectral cell labeling for cell tracing ...

...is an off-target effect. Should I be concerned about off-target effects? Depending on your experimental...this could be a huge concern, or nothing to worry about all. Let’s consider several scenarios below for ...Predicting off-target events As mentioned in the above section, there are gRNA softwares available which...diligence to ensure they are indeed a non-issue. Above we mentioned how to predict off-target frequency...

... (post or pre run in the table above, respectively). Some of the above stains require you to visualize...equivalent to backbone + insert, unsuccessful 2 and 3 above), you can try ligating the recipient plasmid alone...get experimenting!   Additional Resources: Read about Gel Stains on the BitesizeBio Blog  Additional ...

...et al. 2015). Meanwhile, Sanger sequencing is laborious, time consuming and cannot be applied to heterogenous...viable options for multiplex sequencing in many laboratories. Nevertheless, unbiased detection isn’t as sensitive...unbiased sequencing may not be worth the cost and labor when single clones are selected. Conversely, translational... Hough is a Biochemistry PhD candidate in the laboratory of Prof Steve Jackson at the Gurdon Institute...

... “transitivity,” calls into question any claims about specificity of a given RNAi technique. While transitivity...conceptually similar to the microRNA precursor mentioned above and is a popular RNAi trigger in the mammalian research...substantial ways: variable silencing efficiency and labor-intensive processes to isolate RNAi plants. With...resource on the Addgene blog Browse our blog posts about plant biology Check out our CRISPR blog posts Resources...

...mycoplasma contamination in your cell culture or you are about to begin a new cell culture project. If mycoplasmas...that are difficult to trace. These include the laboratory personnel, the serum, the cell culture media,...accounts for the largest source among those mentioned above. The contaminants can spread through dirty clothing...proliferating shortly. Once they are present in the laboratory environment, they are difficult to eradicate....

... infrastructure, and secure collaboration.  In that post, we talked about Addgene’s data processing needs...Last summer, we posted about our bioinformatics quest, which led us to Seqera Labs, a company that provides...

...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...

...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...

...community.  You'll also find tons of information about Addgene's ready-to-use antibodies, viral vectors...and four! So you'll also find Addgenies out and about, attending sessions, visiting posters, and leading...Sunday, from 12 pm - 2 pm. If you want to learn about, and practice, principles of clear, accessible writing...

...scientists using AAV to share practical information about their experiences. It will contain all of the juicy...data to find patterns that we can share with you about promoters, serotypes, expression patterns, and more... more. If you have feedback or suggestions about the AAV Data Hub, we welcome you to share your thoughts...

...supporters of science communication. To help you think about ways to effectively communicate your science, we... of programs SITN runs and learn how they think about communicating science. Listen to the interview ...Interview with Science Rapper Tom McFadden Learn about SITN's work at DayCon 2015 5 Reasons to Use Reddit...

...expedite the application process.  More information about export control licenses is available through the...Industry and Security. If you have any questions about this or other Addgene shipping and ordering processes...customer support team at [email protected].  Curious about how rabies plasmids are used in the lab? Check out...

...researchers time by quickly relaying key points about an unfamiliar topic or lab process. Along with our...goals, or to answer any questions you might have about Addgene. The page is divided into three main sections...a variety of roles at Addgene.  You’ll hear all about their career paths, future goals, and get some useful...

...multiplexing options, but also check out our blog post about multiplexing with Cpf1.   Figure 1: Multiplexing...assembly.  Unlike some of the plasmids described above, this vector does not contain Cas9, so you’ll need...similar to the Csy4 polycistronic system described above, except that they use an endogenous nuclease system...The Proper Cas9 Variant for Your Experiment Learn About Multiplexing with Cpf1 Additional Resources on ...

... Cepko. Listen to the podcast below to hear all about the research being conducted in Professor Cepko'...and mentoring styles. If you'd like to learn more about Professor Cepko's recent work on developing GFP-...the time to talk to us and look forward to hear about more exciting discoveries from her lab. Special ...