...makes generation of high titer Cas9 lentiviruses challenging. This cytotoxicity also causes growth rate variability...Roth’s lab at the University of North Carolina, Chapel Hill. These can be used to investigate the effects...transgene expression across different cell types (Aschauer et al., 2013). Expression of hM4D(Gi)-mCherry ...hM4D(Gi)-mCherry in fixed tissue. References 1. Aschauer DF, Kreuz S, Rumpel S. Analysis of transduction...

...overall positive charge on DNA molecules so that it can more easily cross the negatively charged cell membrane...role in bacterial evolution and is the primary mechanism by which bacteria have gained antibiotic resistance...electroporation, the application of an electrical charge to cells, to increase cell membrane permeability...

... Nat Biotechnol. 2018 PubMed PMID: 30125270. Changing the way we detect viral outbreaks Article contributed...equipment. What’s more, detection is based on color changes to the assay paper, so no special equipment or ...novel peptide ligand motifs Article contributed by Michael Lemieux  Listen to the SH3 domain podcast segment...biochemical processes. One of the largest and best characterized of these is the Src homology 3 (SH3) domain ...

...and γ-retroviral packaging plasmids are not interchangeable. General envelope plasmids, such as VSV-G (... vice versa. Envelope plasmids may be used interchangeably. Are γ-retroviruses replication incompetent...References and resources References Swift S, Lorens J, Achacoso P, Nolan GP. Rapid production of retroviruses ...cell-based systems. Curr Protoc Immunol. 2001 May;Chapter 10:Unit 10.17C. doi: 10.1002/0471142735.im1017cs31...

...Immunologists are coming together to tackle the challenge of curing cancer in cross-disciplinary projects...projects and collaborations. Summarized by Professor Michael Karin from UCSD: “The problem with cancer research...poster sessions During the poster session I got a chance to talk with Dr. Friedmann-Morvinski about tools...27411589. 2. Todoric, Jelena, Laura Antonucci, and Michael Karin. "Targeting inflammation in cancer prevention...

...computation work comes with new sets of challenges. One challenge with computational analyses is to keep... are doing what you are doing. Another common challenge is to keep track of what works, and what does ...that further helps you keep track of your exact changes. Alternatively, you can simply make a note in your...

...s Lab in 2009. From 2009 to today, Sample6 has changed focus and names, swapped leadership, and grown ...much learning, the focus of the application was changed, but Sample6 still leverages the core engineering...his co-founders raised $6 million. The biggest challenge during this time was learning to communicate effectively...better than current technology and get customers to change behavior. When you are ready to commit to the company...

... antibody cross-reacts to, then there is a good chance that the antibody could meet your needs.    Multiplex...will not only have peace of mind as you make your purchase but also someone to turn to for advice if you ...application.      Fig. 1: Antibody Selection Flow Chart    What does validated mean anyway? You now ...Validation!  Download our antibody selection flow chart.  References and Resources Additional Resources...

...monkeys. Surprisingly, we found that this small change made a big difference. Replacing the mCherry with...does highlight the importance of a careful characterization of transgene expression. Which tag should ...RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method...Raper J, Hu X, Paré J, Bonaventura J, Richie CT, Michaelides M, Mueller SAL, Roseboom PH, Oler JA, Kalin NH...

...restriction enzymes or vector purchases (Table 1). Restriction enzymes and vector purchases can raise the cost anywhere...one-off projects, or even save yourself hours by chatting quickly with the postdoc who’s really good at ...harder it is to throw in last minute experiments or changes. Fun fact: Addgene’s high-throughput approach means...

...kits or ordered as synthesis modifications when purchasing your gRNA. Pro tip: modifications are typically...expression systems are constantly “on”. There is no mechanism to turn cutting off or on at a specific time. ...and you can opt to do-it-yourself with a kit or purchase it commercially synthesized with a label. Not ..., E., Satyapertiwi, D., Ramel, MC., et al. Photoswitchable gRNAs for Spatiotemporally Controlled CRSIPR-Cas-based...

... studying protein turnover is radioactive pulse-chase experiments. However, these experiments are problematic...synthesis: incorporation of puromycin into peptide chains." Proceedings of the National Academy of Sciences...PubMed Central PMCID: PMC4414919. 5. Yarmolinsky, Michael B., and L. Gabriel. "Inhibition by puromycin of...Single β-actin mRNA detection in neurons reveals a mechanism for regulating its translatability." Science 343.6169...

...protein homodimerization is characterized by an affinity of 110 µM (Zacharias et al, 2002), meaning that... proteins reported in publications are only characterized under a limited number of conditions, often ...microscopy (University of Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes...

... Joanna Brownstein (our outgoing Chair) and Dave Root (incoming Chair) and our entire supportive Board...year (12% in 2018) since 2005. We’re meeting the challenges of DNA distribution to ensure scientists everywhere...happening. The Addgene team will have some fun challenges this year and I’m excited about this, but definitely...

...and ELISAs. Chromogenic reactions produce a color change, so they are typically used in microscopy-based...coupling can be achieved through several different mechanisms. Most take advantage of chemical groups intrinsically...that make up the disulfide bonds between antibody chains. 3) Endogenous carbohydrates added to antibodies...References Bendall SC, Nolan GP, Roederer M, Chattopadhyay PK (2012) A deep profiler’s guide to cytometry...

...scientists communicate with each other: from formal mechanisms like publishing a research article, presenting...informal settings like lab meetings, Twitter, coffee chats with potential collaborators, blogging and more....read freely by anyone and are typically free of charge for authors to post. They can be assessed and commented...authors over time, providing a public record of changes, critiques or endorsements. In contrast, journal-based...

...CRISPR/Cas9 genome editing can result in unwanted changes at non-target sites. Means to decrease these so-called...between the non-target strand and a positively charged groove formed by the Cas9 HNH and RuvC nuclease...al reasoned that, if they decreased the positive charge in the HNH/RuvC groove, then they could weaken ...interactions between the groove and the negatively charged DNA and therefore destabilize basal strand separation...

... with Cas9 guided by different promoters. The challenge in building genetic circuits is getting enough...shape of the response function or how the output changes as a function of input. Usually that function is...enough genetic parts for complexity. We have characterized almost 600 terminators. That gives us enough... we’ve really been making tons of parts and characterizing the software to help you put it together. The...

...Shyu YJ, Liu H, Deng X, Hu CD. Biotechniques. 2006 Chang-Deng Hu Green sfGFP Split super-folder GFP, our...protein-protein interactions in vivo. Blakeley BD, Chapman AM, McNaughton BR. Mol Biosyst. 2012 Brian McNaughton...Shyu YJ, Liu H, Deng X, Hu CD. Biotechniques. 2006 Chang-Deng Hu Venus Improved N-terminal fragment VN155...signal-to-noise ratio. Kodama Y, Hu CD. Biotechniques. 2010 Chang-Deng Hu mVenus pET-BiFC contains both fragments...

...of the theme of my interview stories as the characteristic which the story demonstrates I possess.   Bullet...to present about your prior research. This is a chance to highlight your prior accomplishments and demonstrate...carefully. And even if you don’t have technical challenges, your interviewers might.  You’ll probably still...their hand to ask a question or type it into the chat. Either way, just let the interviewers know up front...

...experiments are either not accessible or too challenging for amateur scientists. Planarians, wormy creatures...cell types and for viewing how gene expression changes through time or after genetic manipulation. The...tissue sample, several washes, and temperature changes to visualize the expression of specific genes. ...genes of interest and was first discovered and characterized in an unrelated worm (C. elegans) [2]. Delivering...

...Indicate the amino acid change as well as any functional consequences of that change. For example, “Mutated... Answer “What does this plasmid do?” in <200 characters. A descriptive purpose focuses on the practical...Addgene Blog Find Articles Citing Addgene Plasmids Searchable and Sortable gRNAs for Your Next CRISPR Experiment...

...tasks, both of which are factors in the technical challenges they can present. Many researchers find themselves... following transfection. In other words, when exchanging the medium after transfecting cells, replace ...electrostatic forces between these two negatively-charged membranes (Davis et al., 2002). Polybrene is one...306. PubMed PMID: 10634178. 8. Murnane, John P., Michael J. Yezzi, and Barbara R. Young. "Recombination ...

...light-activated ion channels. Similarly, chemogenetic tools are designed channels that are chemically ...introduced in neurons and depending on the choice of channel protein, they can trigger neuronal excitation or...adeno-associated virus. Curr Protoc Microbiol. (2013); Chapter 14:Unit14D.5. PubMed PMID: 23686825. PubMed Central...

...the only right way, and Indecisives, who keep changing their minds and have trouble making decisions.... of your supervisor’s management style, bring challenges to his or her attention as soon as you can. It... action items. This will give your supervisor a chance to review what you have discussed and add suggestions...mutually beneficial solutions. If you package your challenges with your supervisor into learning opportunities...

...genomes, cloning vectors, and helper plasmids (Chadeuf et al., 2005, Wright et al., 2011). In fact, the... software. References and resources References Chadeuf G, Ciron C, Moullier P, Salvetti A (2005) Evidence...Redon R, Moullier P, Léger A (2015) Advanced Characterization of DNA Molecules in rAAV Vector Preparations....2015.32 Wright JF, Zelenaia O (2011) Vector Characterization Methods for Quality Control Testing of Recombinant...

...AAV-containing 40% fraction is collected and buffer exchanged to remove the iodixanol and concentrate the purified...or the virus can be stored at 4 °C and buffer exchanged the next day. Refer to the AAV Iodixanol Gradient...correctly. When aliquoting purified and buffer exchanged virus, remember to make one or two small aliquots...vivo use. Current protocols in molecular biology, Chapter 16, Unit 16.25. PubMed PMID: 18265393. Additional...

...titration by qPCR.   Pro tip! An optional media exchange can be performed 12–18 hours post-transfection...the density gradient material by dialysis/buffer exchange and further concentrate the virions if needed....Lock, M., McGorray, S., Auricchio, A., Ayuso, E., Beecham, E. J., Blouin-Tavel, V., Bosch, F., Bose, M., ...., Francis, J. D., … Snyder, R. O. (2010). Characterization of a recombinant adeno-associated virus type...

... 37 °C and 5% CO2 and get agitated with sudden changes in temperature (especially if you have air conditioning... to recover after they are passaged. This could change their growth rates and morphology, rendering their...numbers Keep an eye on the passage number. Cells change their growth rate and morphology at high passage...Additional Resources on the Addgene Blog The Challenges of Cell Culture Cloning Mammalian Cells with ...

...through wild populations. Whereas most genomic changes impose a fitness cost and are eliminated by natural...previous gene drive and thereby undo the genetic changes driven by the earlier intervention2. While an initial.... PubMed. Gantz, V. M. & Bier, E. The mutagenic chain reaction: A method for converting heterozygous to...10.1126/science.aaa5945. PubMed. DiCarlo, J. E., Chavez, A., Dietz, S. L., Esvelt, K. M. & Church, G. M...

...purify proteins can face any number of technical challenges depending on their protein of interest. After...After several decades of trying to address these challenges, researchers have amassed a considerable molecular...for your protein. If you’re working with an uncharacterized protein, or a protein for which a good antibody...KETAAAKFERQHMDS  1.8 Solubility and Affinity Abundance of charged and polar residues improves solubility; good for...

...following changes. In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed the replicon...same year. Her research is focused on the escape mechanism of genome editing in microorganisms which could...cells to understand the genetic and biochemical mechanisms behind their distinct phenotypes and to design...

...Design). This modular approach combines an interchangeable set of fusogenic and targeting components to...where the fusion and targeting components are interchangeable. Image from Strebinger et al., 2023, reused...local proteome, but some applications have been challenging or inaccessible. The Ting lab recently made two...They demonstrated LOV-Turbo in neurons, in pulse-chase experiments, and more. Find LOV-Turbo plasmids here...

...the chances of a cell being infected by only one viral particle, but it also increases the chances that...presentation deceive you; pooled libraries can be quite challenging to use! In this post, I’ll walk you through how...virus. Let’s start there!     Figure 1: Flowchart showing the process for using pooled lentiviral...

...General Meeting seven other times I had subsequently changed fields and my cohort of friends and colleagues ...evolutionary lineages share genes that can improve chances of survival for an individual, and eventually, ... cells regulate gene expression in response to changes in cell density. At the mixer, they meet other ...Dill-McFarland, Lisa Gorski, Mark O. Martin, Maureen Berg, Michael D. L. Johnson, Tiffany Lowe-Power, Eric Griffis...

...of reprogramming (or “Yamanaka”) factors, they changed the landscape of regenerative medicine. Their work...viral vectors are preferred. This is to avoid the chances of insertional mutagenesis, which is the integration...replacing C-MYC with L-MYC [10, 11]. While the mechanisms underlying the enhancement of iPSC generation...Induced Pluripotent Stem Cells (iPSCs): Benefits, Challenges and the Road Ahead." Journal of Alzheimer's disease...

...expertise with that of the company. To increase your chances at successfully applying for a position, you should... in a technical support role. What general characteristics do jobs in technical support have? There are... less time consuming and/or less technically challenging. Alongside there is a second-line team that takes... ways to communicate with customers. Online live chat is a convenient alternative for asking a quick question...

...incentivize good mentorship: AMA mechanics: Be prepared! The mechanics of doing the AMA were interesting...Getting on a good science career path can be challenging these days, but for many of us, scientist is ...Science through Video with the Addgene Youtube Channel Check Out Vidoes of Gender & Science from LabLinks...

...effects. The flaws of RNAi screens are well characterized and strategies exist to control for these faults...also reproduced several previously observed characteristics that influence gRNA off-target activity: 1)...23394947. Pubmed Central PMCID: 4299491. 5. Bassik, Michael C., et al. "A systematic mammalian genetic interaction...available from Addgene Other Resources Watch Dr. Michael Bassik’s lecture on “Multiplexing with CRISPR Screens...

...protected by Cas9. This is similar to the proposed mechanism for engineered pegRNAs (epegRNAs), which add a...of mouse monoclonal antibodies generated and characterized by the Geiss Lab in their effort to fill a void...cells. Plus, Voltron2’s fluorescence is tunable by changing the dye that becomes conjugated with its HaloTag...gene expression regulation, enhancing both the mechanisms of delivery and the potential for therapeutic...