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We narrowed to 810 results for: nin

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  1. A Needle in a Base-Stack: Cas9 Structural Biology

    Type
    Blog Post
    Published
    June 4, 2024, 1:15 p.m.
    ...species, explaining why they recognize different guide RNAs. Deep in the REC lobe, the arginine-rich bridge... we can see why: two arginines make base-specific hydrogen bonds to the guanines, but the N (a thymine...guide RNA is bound primarily by the REC lobe. An arginine-rich bridge helix connects the two lobes and helps...stem loops bind along the outside of the protein, spanning both lobes. Stem loop 1, nestled between the REC... (yellow) make base-specific hydrogen bonds to guanines of the PAM in the non-target DNA strand (dark ..., 2022; Sternberg et al., 2015). The correct positioning of the non-target DNA strand is important for...DNA strand, and Cas9 bends the DNA helix by transitioning between the open and closed conformations. This...

  2. New CRISPR Tools: Cas7-11 and PASTE

    Type
    Blog Post
    Published
    Feb. 17, 2022, 2 p.m.
    ... revolutionary discovery, including CRISPR, is turning potential into impact. In molecular and cellular... contradicting their initial assumption. Eugene Koonin and Kira Makarova, longtime collaborators of Omar... the project. Using computational observations, Koonin and Makarova found that Cas7-11 appeared to have...toxicity compared to other RNA editing methods — streamlining experimental design and its potential for clinical...reported no detectable effect on cell viability – meaning there was no measurable cell toxicity – using Cas7...integration was observed in non-dividing cells, meaning that this system wasn’t dependent on the cell’s...sequence and integrate it into any DNA sequence containing the 38-50 bp sequence recognized by the serine...

  3. Viral Production at Addgene

    Type
    Blog Post
    Published
    Oct. 24, 2023, 1:15 p.m.
    ...electrophoresis (PAGE) followed by silver staining or SYPRO Ruby staining and the molecular weight and relative...after negative staining. Empty vector particles can be identified after negative staining and appear darker...of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV subjected to ...Microscopy The ratio of empty to full (i.e., genome-containing) AAV particles within representative vector preparations... Trono lab. Cell culture medium (OptiPro SFM) containing lentivirus is first cleared by low-speed centrifugation...followed by centrifugation. Precipitated pellets containing viral particles are then resuspended in PBS, ...

  4. New Acoustic Reporter Genes: Ultrasound Imaging of Gene Expression

    Type
    Blog Post
    Published
    Jan. 24, 2023, 2:15 p.m.
    ...extract, and 5 g/L NaCl) or on LB-agar plates containing glucose and L-arabinose at 37oC for at least ...induced with arabinose and colonies on arabinose-containing plates will be visibly white, and gas vesicles...expression heterogeneity required single cell screening to isolate a clonal cell line capable of producing... gene silencing required addition of chromatic-opening agents to boost expression to appreciable levels...Anabaena GVs in mammalian cells without the shell stiffening protein GvpC, which allowed the GVs to be flexible...vesicle expression and robustness without chromatin-opening agents. It ended up being very straightforward ...cells for a several generations to dilute the unintegrated plasmids (Fig. 2b). Next, perform a short (12...

  5. Deciphering the Mysteries of Behavior with Viral Vectors

    Type
    Blog Post
    Published
    Sept. 17, 2019, 12:40 p.m.
    ...recordings and lesioning studies have been essential for this task. However, most lesioning methods destroy...destroy areas larger than intended and may unintentionally remove connecting pathways between other distant...function, with far fewer side effects compared to lesioning methods -  they may be the key to further unraveling... brain areas associated) compared to an animal running to escape a threat. Locomotion can thus be used...transgenic animals and viral vectors, scientists are combining genetic manipulations with the forced swim test... as sleep, food intake, mobility/coordination, learning, and memory. We’ll cover these in a  future blog... Vincent Jean Pascoli, and Christian Lüscher. "Refining deep brain stimulation to emulate optogenetic ...

  6. Bioinformatics at Addgene

    Type
    Blog Post
    Published
    July 14, 2022, 1:15 p.m.
    ... process and tested it by running the main Nextflow script file (containing our new process) on our local...From the beginning, one of Addgene’s goals was to ensure the scientific community obtained high-quality...pipeline. Benefits of Nextflow for bioinformatics pipelining In many use-cases, including ours at Addgene,...a container is sequestered from other programs running on a computer, so it safely operates in isolation... to the ease of jumping back and forth between running the pipeline locally and in the cloud. Creating...print statement at the end of the script or by opening the files. By working on one process at a time,...and decreased cost compared to purchasing and maintaining our own infrastructure. Our patchwork collection...

  7. Our Most Popular Blog Posts from 2019

    Type
    Blog Post
    Published
    Dec. 31, 2019, 2:26 p.m.
    ... and find plasmids containing these fluorescent proteins at Addgene. 7. Designing Your Chalk Talk for... Hot Plasmids - March 2019 - Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable...Proteins by Cary Valley, Shreya Vedantam, Michelle Cronin, and Angela Abitua Thanks to a team effort from...101: Positive and Negative Selection for Plasmid Cloning by Jennifer Tsang There are many strategies for...systems and sacB counter-selection) tools used in cloning. 8. Bright Monomeric Fluorescent Proteins: mNeonGreen...chalk talk for an academic job interview including defining your research vision, organizing your vision into...

  8. CRISPR Kinome Libraries Available: Pooled and Individual Plasmid Formats

    Type
    Blog Post
    Published
    Sept. 9, 2016, 2:43 p.m.
    ...at the Broad Institute, John Doench. A genetic screening project can be a tremendous undertaking, producing...sad. Learn More About CRISPR Pooled Library Screening Why the kinome? While properly designed perturbations...technology, gRNAs to knockout genes – are essential screening components, the model system and the assay are.... The individual plasmids will be useful when screening for phenotypes, such as cell-non-autonomous effects...effects, that do not lend themselves to screening in a pooled setting. The individual plasmids will also...imaging or gene expression assays necessitating the screening of individual plasmids separately. Additionally... will find these reagents to be useful. Happy screening! Many thanks to our guest blogger, John Doench...

  9. Working 9-5: an Addgene Co-op

    Type
    Blog Post
    Published
    June 2, 2022, 2:15 p.m.
    ...provided with a detailed training schedule that helped me transition easily. Training and responsibilities...responsibilities Through this training, I was gradually introduced to Addgene as a company and given more details on...on what my position entailed. The official training process was lined out to span the first three months...months of my six month employment. In the beginning, I spent a lot of time shadowing members of my team ... what I was to do for my time at Addgene. This training prepared me for the independent work on the QC...concern to the plasmid function. The steps of the training process gave me enough time to become familiar...

  10. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    Published
    Sept. 5, 2017, 1:58 p.m.
    ...commonly, DSBs are repaired by the nonhomologous end-joining (NHEJ) pathway, leading to nonspecific nucleotide...Furthermore (3), we have developed a framework that, by combining these two strategies allows you to specifically...find the 0.2-0.5% correctly edited alleles, when screening reasonable amounts of single cell clones. Furthermore...Furthermore, CRISPR/Cas9 editing is often bi-allelic, meaning that even if one allele is accurately edited, the... few hundred. From these two steps, you’ll be screening a total of a few hundred clones - Still better...longer feasible to incorporate mutations without screening thousands of clones to find a positive one. So... single cell. We calculated the likelihood of obtaining homo- and heterozygous mutations by multiplying...

  11. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

    Type
    Blog Post
    Published
    April 24, 2015, 2:39 p.m.
    ...that there was no cross-reactivity, opening the door for immunostaining analysis. To achieve even cell labeling..., but can be detected via immunostaining. This feature facilitates screening of Cre-negative Brainbow ...systems. Pre-Brainbow techniques, such as Golgi staining, injection of diffusible labels, or electron microscopy...segment, eliminating two color possibilities. The remaining cassette can invert as long as Cre remains expressed...Second, the system did not permit analysis by immunostaining. Although the fluorophores used are fluorescently... Cai et al. also created an Autobow construct containing both Cre and XFPs. Cre production drives recombination...

  12. CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9

    Type
    Blog Post
    Published
    Oct. 11, 2016, 2:30 p.m.
    ...Agrobacterium-compatible, Cas9 containing vectors ready for Golden Gate mediated cloning of your gRNA of interest...researchers to insert targeting oligos with minimal cloning required. As with other model systems, multiple...promoters (Belhaj et al., 2013). Addgene carries Cas9-containing plasmids for knockout, activation and repression...on the helper plasmid and used to insert the intervening region into the plant genome. Created with BioRender.com...deaminase-mediated base editing (cytosine base editor or adenine base editor) and reverse transcriptase-mediated... et al., 2019). Both cytosine base editors and adenine base editors have been developed for plants and...Demirer GS, Zhang H, Ye T, Goh NS, Aditham AJ, Cunningham FJ, Fan C, Landry MP (2019) DNA nanostructures...

  13. Antibodies 101: Beyond Surface Labeling

    Type
    Blog Post
    Published
    June 27, 2024, 1:15 p.m.
    ...will introduce you to a few useful labeling and staining techniques beyond standard surface labeling. Pro... Pro tip! Many people use 'labeling' and 'staining' interchangeably to refer to any technique that combines...Addgene uses 'labeling' to refer to antibodies and 'staining' to refer to small molecule dyes. Indirect labeling...labeling must be performed only after viability staining and surface labeling are already done. Intracellular... viability. Unlike intracellular labeling, dye staining can be done on live cells. For cellular proliferation...cell, the ester group is cleaved off by enzymes, turning the resulting molecule fluorescent and membrane-impermeant... for specific downstream applications, while maintaining a clean cell surface, dump gating is a useful...

  14. Transferable Skills Guide: Conflict Resolution

    Type
    Blog Post
    Published
    Jan. 4, 2022, 2:15 p.m.
    ...alleviate those differences, they had a hard time maintaining their friendships. I put “neutral” in quotations...learn how to sell them. Types of Conflict Before explaining my approach to conflict resolution, it’s important... into a gap between teams that nobody is really owning. Conflict in this latter scenario usually manifests...disagreeable, you recognize their feelings as valid. Listening: In one of my favorite books, Michael Ende’s Momo... nearby village (and to the reader) by simply listening. Chances are, you know someone like this, even...conflicts.  Institutional Knowledge: Of the tools I’m mentioning here, this may be the most controversial, so ...also aggravate long-term personal conflicts.  Transitioning a conflict into a collaboration may not be the...

  15. Advice for Choosing a Research Project

    Type
    Blog Post
    Published
    April 17, 2014, 2:45 p.m.
    ...their goal of PhD training when selecting a research topic. The end point of training is not to become ...After joining a lab, the focus shifts to choosing an unexplored and impactful research topic that aligns...consider when choosing a research project After joining a lab, the next important decision to make is “...what approach will I take and what can I learn, learning the difference between good and bad data, and ... dreams. He also highlights the importance of combining “new discoveries or technologies increase chances...Advice for Choosing a Research Project Finding and Joining Your Dream Lab Advice for Choosing a Research Project...

  16. Walking beside Academia and giving it a high five: My transition from graduate work to a position at Addgene

    Type
    Blog Post
    Published
    July 2, 2015, 8:37 p.m.
    ...demonstrates that transitioning from academia doesn’t have to mean abandoning it or running away screaming...weren’t some pretty hefty bumps during my graduate training. I particularly think that too many young scientists...talking to people about science and that I love explaining things to people. When I started my graduate ...keeps me on top of a variety of fields thereby broadening my knowledge. In addition, this position allows...careers outside of academia during my graduate training. PhDs have many roles to play inside, on the outskirts...

  17. Choosing a Good Mentor for Scientists

    Type
    Blog Post
    Published
    Jan. 16, 2014, 2:56 p.m.
    ...scientist-in-training will spend 10 or more years with a small number of formal advisors learning how to be...priorities will help in determining a good fit. Here are just a few suggestions for determining whether an advisor... will be ones that help them succeed after the training period. While there are many aspects to choosing...time hanging out with members of the lab. It is a warning sign if the PI does not welcome you to visit and...Precision and Plasticity in Mentoring" Don’t ignore warning signs If lab members, neighbors or alumni give ...

  18. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    Published
    March 8, 2021, 2:41 p.m.
    ...for staining. To address this, the membranes are incubated before staining in a buffer containing nonfat...in the presence of a reducing agent, typically containing thiols, to cleave disulfide bonds. Load the samples...variety of tissues to demonstrate that the protein staining matches the expected expression patterns across...level of validation ensures that the antibody is staining the intended target.  Once you have chosen an ...used by the vendor for validation could contain unintended cross-reactive proteins. Similarly, if you vary...with a good overview of the western blot. When planning a western, take care to include the proper positive...

  19. Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens

    Type
    Blog Post
    Published
    Feb. 24, 2015, 7:50 p.m.
    ...the next step in high-throughput, genome-wide screening,” Konermann said. “If you want to activate all...her colleagues demonstrated the new system by screening for genes that, when turned on, make melanoma ...CRISPR library enabling genome-scale, knockout screening). As the Zhang lab's Nature paper reports, “Here... can facilitate genome-scale gain-of-function screening when combined with a compact pooled sgRNA library...validation of the top 10 hits.” Two-way CRISPR screening In October, Jonathan Weissman’s lab introduced...developing sgRNAs that will enable even more fine-tuning of gene expression levels and on software for analyzing...Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann M, Weissman...

  20. 15 Years of Addgene: The Top 15 Plasmids

    Type
    Blog Post
    Published
    Jan. 8, 2019, 1:54 p.m.
    ...and cloning sites for inserting the guide sequence (pSpCas9(BB)). The 2A-GFP allows for screening of transfected.....From moving office locations three times, to opening our UK office in 2014, to starting our viral vector...virus, making it ideal for CRISPR pooled library screening experiments. Find lentiCRISPR v2.(If you’ve peeked...  Find the scrambled shRNA vector. pLKO.1 - TRC cloning vector - This vector from the David Root lab is...3rd generation lentiviral transfer plasmid for cloning and expressing new shRNA sequences. For more information...slowly than standard plasmids. Find pLKO.1 - TRC cloning vector. Tet-pLKO-puro - Tet-pLKO-puro contains ...

  21. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

    Type
    Blog Post
    Published
    May 26, 2020, 1:15 p.m.
    ...mutated methionine was not enough to prevent alternative initiation, so they mutated the methionines at position... You can use DNA sequencing or other molecular cloning techniques to determine CRISPR/sgRNA efficiency...homology directed repair (HDR) and non-homologous end joining (NHEJ). In these reporters, editing typically results...H2B-GFP. This visual read out allows for easy screening of different gRNAs by simply changing the gRNA...transfected cells will express RFP while cells containing the reporter gene that was cleaved by Cas9 and...start site initiation, the lab removed the native methionine start site. This first iteration was named GEmCherry1...

  22. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    Published
    March 21, 2017, 2:30 p.m.
    ...consisted of two plasmids: the packaging plasmid containing all the structural genes necessary for the production...retrovirus into the packaging cell line PA12 (containing the psi deletion). A double crossover event between... LTRs, the ψ sequence, a part of the env gene containing the rev response element (RRE), an internal promoter...transfer vector plasmid), 2) the HIV-1 genome containing all viral genes with the exception of the env...distinct expression vectors to target cells - one containing the gene of interest (pTet-IRES-EGFP, pPRIME-...bottleneck for your experiment. Single vector systems containing both your transgene and tTA/rtTA (pINDUCER20,...your institution’s Biosafety Committee before beginning any work. Lentiviral production and transduction...

  23. Mycoplasma Contamination: Where Does It Come From and How to Prevent It

    Type
    Blog Post
    Published
    Oct. 8, 2020, 1:15 p.m.
    ... the cellular DNA. Fluorescent DNA staining is another DNA staining alternative. This method uses DNA ...proper training and guidance to the people working in the lab facility. This can be done by training eligible...this task, you can use PCR based detection, DNA staining, fluorescent tagging, agar plating, etc. Be wise... is added to the liquid or semi-solid medium (containing nutrients essential for mycoplasma growth). Infected...facility periodically. The CO2 incubators, used for maintaining cell lines, must not be overcrowded (filled no...contamination. Be aware and cautious rather than learning it the hard way! Many thanks to guest blogger...

  24. The Future of Research Symposium Boston 2015

    Type
    Blog Post
    Published
    Oct. 20, 2015, 2:30 p.m.
    ... the tides may be turning. In 2013, the NIH announced its first round of Broadening Experience in Scientific...achieved tenure-track positions within 5-7 years of earning their PhDs (3), showing that the ‘standard’ pathway...Scientific Training (BEST) awards, which are designed to support new approaches that address the need to... School. Graduate education programs are also beginning to address these issues independently; for example... may look like. The panel discussion on Friday morning will be dedicated to Academic data and the Labor...PMC3342243. 4. Mathur, Ambika, et al. "Transforming training to reflect the workforce." Science Translational...

  25. Transferable Skills Guide: Problem Solving

    Type
    Blog Post
    Published
    Nov. 30, 2021, 2:15 p.m.
    ...found that many researchers feel they lack formal training in a variety of transferable skills. At Addgene...puzzling through your broader research questions and designing future experiments. Being able to work through...at work.   While I was in graduate school and beginning to research non-academic careers, I always felt...dozens of career advice articles, I settled on defining problem solving skills as: One’s ability to logically...the process, you might need to go back to the beginning, reassess the situation, and try a new strategy...scientific method is a core part of our academic training.  Developing strong problem solving skills is ...challenges we encounter. As you spend time in a lab learning techniques and taking on independent projects,...

  26. The time and cost required to make a plasmid

    Type
    Blog Post
    Published
    March 23, 2023, 1:15 p.m.
    ... a plasmid? Or how much time you have to spend cloning before you can start your experiment? What about...order? Sometimes, it feels like an eternity of cloning, waiting, and repeating before you can finally ...course, these were all 2018 numbers, and the intervening five years, inflation has occurred. Here at Addgene...in science – the time spent by the scientists designing and performing the experiments. This cost varies...and money across the board. Consider alternative cloning strategies There are many strategies beyond the...well) plate! How much do repositories help?   Designing precise calculations for any of the numbers in...Addgene Blog 5 factors to help you choose the right cloning method Find blog posts about scientific sharing...

  27. It’s Bioelectric! An Exciting Interdisciplinary Field of Research

    Type
    Blog Post
    Published
    Oct. 29, 2019, 2:52 p.m.
    ...can also affect patterning and in some cases even override genetically encoded patterning. Based upon trends... Bioelectric states can alter cell types and patterning There is mounting evidence that signaling via... even be necessary and sufficient for certain patterning activities of entire tissue types or even organs...Since bioelectrical signals are controlled by the opening and closing of ion channel proteins, they are not...developmental biologists and cancer researchers are beginning to turn their attention to bioelectricity. One...Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis." Development 139.2 (2012):...

  28. Channelrhodopsins with improved light sensitivity for minimally-invasive optogenetics

    Type
    Blog Post
    Published
    Nov. 3, 2020, 2:15 p.m.
    ...channelrhodopsin-expressing neurons, the channelrhodopsins respond by opening and allowing an influx of ions into the neuron ...s lab and Viviana Gradinaru’s lab used machine learning to design new channelrhodopsins that are more ...channelrhodopsin that’s successful in vivo. Using machine-learning to overcome channelrhodopsin design challenges... Arnold and Gradinaru Labs developed a machine-learning model to help them construct “designer” channelrhodopsins...the library to 1,161 sequences. Thirty of these remaining sequences were selected for characterization with...mice that received ChR2(H134R) did not turn. Left-turning behavior in ChRger2-treated mice ceased when the...Mackey ED, Gradinaru V, Arnold FH (2019) Machine learning-guided channelrhodopsin engineering enables minimally...

  29. Illuminating Choices: A Guide to Selecting Fluorescent Dyes and Ligands

    Type
    Blog Post
    Published
    March 21, 2024, 1:15 p.m.
    ...visualization of both organic tissues and cell cultures, opening biological interiors to many inquisitive scientists...select the optimal dye or fluorescent ligand when designing an experiment. You may find yourself defaulting...Fluorescent dyes utilize these fluorophores by combining them with a molecular species with high affinity...affinity for an endogenous molecular target, meaning the dye will be attached to the target through binding...so extra care must be taken to assess if proper staining is achieved. Fluorescent ligands have more flexibility...) can be stably combined with nanoparticles, maintaining intensity at ~85% of its original value after...brightness and high degree of overlap. Dyes for DNA/RNA staining should have high affinity and specificity to nucleic...

  30. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    Published
    March 8, 2017, 3:15 p.m.
    ...vector”. Cloning Method Choose from: Gateway cloning, gene synthesis, Gibson cloning, ligation-independent...ligation-independent cloning, restriction enzyme, TOPO cloning, unknown, or other. Bacterial Resistance Enter...possible, we propagate plasmids in the standard cloning strain DH5α. For plasmids with highly repetitive...plasmids, choose the NEB Stable strain. For plasmids containing the ccdB gene, such as Gateway vectors, choose...

  31. Mouse Modeling, Part 1: Genetically Engineered Mice

    Type
    Blog Post
    Published
    July 11, 2019, 1:26 p.m.
    ...maximum genetic heterozygosity in the population, meaning they more closely mimic the genetic diversity of...be engineered by inserting a targeting vector containing the gene of interest into a zygote, or into embryonic...microinjection of DNA or infection with a viral vector containing the gene construct can be used to insert the ...knockin mice. Co-injection of a targeting vector containing homologous ends to the breakpoint promotes homologous...engineering favors DNA repair via non-homologous end joining, the newly inserted gene may have errors introduced...creating the floxed gene segment. Crossing of mice containing a floxed gene to a Cre-expressing strain (which...post of this series, I’ll discuss crossing and maintaining a mouse strain. Additional resources on the...

  32. Viral Vectors 101: Chemogenetics

    Type
    Blog Post
    Published
    July 11, 2023, 1:15 p.m.
    ...combined with. Combining a PSAM with the IPD of a cation channel, such as the serotonin 3 receptor, will...understand the complex wiring of the brain and the underpinnings of neurological disease, neuroscientists need...a popular tool in the field as it provides a noninvasive method for manipulating cell activity, especially...provided by chemogenetics are instead that it is noninvasive and has longer-lasting effects. Building your...issues can be mitigated by using low doses and running appropriate controls (Jendryka et al., 2019), they...promote depolarization in neurons. Alternatively, combining with the IPD of an anion channel, such as the ...

  33. Meet an Addgenie: Insights into Grad School, Career Paths, and More

    Type
    Blog Post
    Published
    June 25, 2020, 1:15 p.m.
    ..., I have conducted informational interviews with nine Addgenies. There are a few patterns throughout the... Graduate school: Do I or don’t I? I asked the nine Addgenies about why they did or did not go to grad... had always been a student and I truly enjoyed learning.” Addgene’s Director of Product Management Eric...feeling like I could read a scientific paper from beginning to end and really get it.” On the other hand, ...don’t get your PhD you’re set up for failure. Listening to Kenkel’s story has helped me realize that is... see where my life will take me. I’m also just learning. I have a huge interest in data science,” which...they work at Addgene. I posed this question to all nine of my interviewees. Fabienne Furt, Addgene’s Associate...

  34. Light Sheet Fluorescence Microscopy

    Type
    Blog Post
    Published
    July 16, 2015, 2:30 p.m.
    ...Neurological Surgery at the University of Miami. The beginning of this century has seen some major advances in...originally named orthogonal-plane fluorescence optical sectioning (OPFOS) (2). Arne Voie, David Burns and Francis...confocal microscopy In confocal microscopes, optical sectioning of a specimen is based on discriminating the ...to photobleaching.  Furthermore, point-by-point scanning along the entire specimen makes imaging too slow...photo-bleaching or photo-damage than in conventional laser scanning microscopy . Since image acquisition is performed... of imaging rodent brains and spinal cords by combining Ultramicroscopy with tetrahydrofuran-based tissue...Leischner, A. Schierloh, N. Jährling, C.P. Mauch,  K. Deininger, et al. (2007). Nature Methods, 4, 331–336. Pubmed...

  35. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    Published
    Oct. 23, 2018, 11:49 a.m.
    ... nearly every experiment requiring AAV. Before joining my lab, I had never worked with AAV, so naturally... Two to five days after transfection, the AAV-containing cells and media are harvested and purified. In...duplicated regions like the ITRs. Restriction digest screening for ITR deletion is still necessary. To save time...overlaid. Following ultracentrifugation, the AAV-containing 40% fraction is collected and buffer exchanged...titering by qPCR quantifies the number of genome-containing viral particles that are present in an AAV prep...standard curve, which is generated from an ITR-containing plasmid. This whole process takes ~3 hours to...expression in mammals Use the PEI calculator for planning AAV packaging transfections Find tips for a first...

  36. Hot Plasmids and Viral Preps - May 2021

    Type
    Blog Post
    Published
    May 18, 2021, 1:15 p.m.
    ...were used in this study.  The zCas9i cloning kit is a plant cloning kit that contains a series of vectors...Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz As multiple antibody-based vaccines...cross-reactivity, antibody candidates underwent affinity screening to select for slow off-rate binders. The best ...protein VP1 is co-expressed with a fusion protein containing VP2C and a protein of interest (POI). The VP2C...with more on the way. S5E2 regulatory element containing vectors developed in the Jordane Dimidschsteind...provide expression selectively in Parvalbumin containing cortical interneurons. We now provide ready-to-use...

  37. 10 Basic tips for mammalian cell culture

    Type
    Blog Post
    Published
    July 12, 2018, 1:09 p.m.
    ...you should always ensure nothing is covering it. Turning on the UV light 3-4 times a week for up to half...microbe-laden aerosols that may enter the hood. Planning your experiments thoroughly before entering the...solves most problems before they occur. With proper planning, you’ll know exactly what materials you’ll need...changes in temperature (especially if you have air conditioning on) so leave them in a sterile incubator. This...make up your master stock, while you can use the remaining vial to grow and freeze additional cells that ... Universität Berlin, Germany. She will soon be joining her PhD lab at the Max Planck Institute of Colloids...the Addgene Blog The Challenges of Cell Culture Cloning Mammalian Cells with the Agarose Method Plasmids...

  38. Negotiating Work and Life: How to Find the Joy

    Type
    Blog Post
    Published
    March 20, 2014, 3:04 p.m.
    ... on the Society for Laboratory Automation and Screening (SLAS) Electronic Laboratory Neighborhood.   Work...If you enjoy working and come home happy in the evening, your friends and family will truly sense and appreciate...within these bounds You can work later (in the evening, or in a few years) when life responsibilities ...Someone else can clean your house, pick up your dry cleaning, cook your dinner and deliver your groceries. ...nights when you can’t get home, babysitting or running errands. Give help when you have time, and in return... things can’t. I kiss my kids good night every evening I am home no matter how busy I am. Believe it or... really busy, but I go to the gym almost every morning. Of course, this is more than just fun, as your...

  39. New Optimized Genome-wide CRISPRko, CRISPRi, and CRISPRa Libraries

    Type
    Blog Post
    Published
    Oct. 4, 2018, 12:44 p.m.
    ...loss of function meaning that if an essential gene plays a role in the desired screening phenotype, it may...knock out, inhibit or activate target genes by combining specific sgRNAs with Cas9 or Cas9 derivatives....copy number dsDNA cutting. A Dolcetto library containing only 3 sgRNAs (setA) also outperformed CRISPRi...conducted with only two constructs (the other containing dCas9-VP16). Like CRISPRi, CRISPRa is only effective...the SAM library approach in a comparison study screening for vemurafenib resistance in A375 cells. The ...screens Read this blog post about genome-wide screening using CRISPR Resources on Addgene.org Find CRISPR...

  40. Plasmids 101: Choosing an Antibiotic Resistance Gene

    Type
    Blog Post
    Published
    Oct. 10, 2023, 1 p.m.
    ...so that you can specifically isolate bacteria containing your constructs of interest. But does it matter...will lose it during division. These non-plasmid-containing bacteria can compete with and confound the goal...from forming while effectively selecting plasmid-containing bacteria. So… why do some people still use amp...? Zeocin is genotoxic when not 100% inhibited, meaning it will induce mutations in the host/plasmid DNA...Excellent No Bacteria, plants, and eukaryotes Not Tn5-containing cells {% icon icon_set="fontawesome-5.14.0" name...References and Resources References Hwang, J. M., Piccinini, T. E., Lammel, C. J., Hadley, W. K., & Brooks...Antibiotic Resistance Plasmids 101: Blue-White Screening Choosing Your Perfect Plasmid Backbone  ...

  41. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    Published
    Aug. 9, 2016, 2:30 p.m.
    ...research interests include proteomics, machine learning, and visualization for big biology. GPS for the...1, 10) indicates a stretch of DNA at the very beginning of the third chromosome, ten base pairs in length... us to transform raw genomic coordinates into meaningful experiments. Table 1: An emerging family of "...all of those 13,000 regions are important for maintaining ESCs. So which of these 13,000 regions are critical...enhancers! This leads us to a three-step approach for designing an experiment to identify the critical NANOG binding...Lentivirus from Addgene If you’re interested in designing your own CRISPR screens for enhancers, check out...database. Aneesh's research interests span machine learning, proteomics, and user interfaces for math.     ...

  42. Tips for Writing a Good Cover Letter

    Type
    Blog Post
    Published
    May 21, 2024, 1:30 p.m.
    ...letter, they have an opening, a body, and a closing. Parts of a cover letter The opening paragraph is the... by giving specific examples of your work and explaining how your experience and skills would help you...application. These could be logistical, like briefly explaining a gap in your job history or sharing relocation...your organizational system, your rationale for designing it the way you did, and state that it would help...how you feel connected to their work. Since the beginning is often the hardest part, here are some examples... in [specific topic] and in the possibility of joining your team as a postdoctoral researcher. Dear Hiring...

  43. AAV Titers: Where Do They Come From And What Do They Mean?

    Type
    Blog Post
    Published
    Nov. 15, 2017, 3 p.m.
    ...after negative staining. Empty vector particles can be identified after negative staining and appear darker...Physical titer: the concentration of viral particles containing viral genomes. Physical titers are measured by...full to empty viral capsids: the ratio of genome-containing viral particles relative to the total number ...approximately 50% of viral capsids were genome-containing (“full”) (Zeltner et al., 2010), while another...Grimm et al., 1999). The percentage of genome-containing viral capsids is typically quantified by electron...representative viral vector preparations are genome-containing (Figure 1).    Figure 1: Electron micrograph...

  44. How Exercise Purges Stress and Keeps Depression at Bay

    Type
    Blog Post
    Published
    Nov. 25, 2014, 4:02 p.m.
    ...muscle expression of kynurenine aminotransferases, to enhance the conversion of kynurenine into kynurenic acid...muscles led to another clue: differences in the kynurenine pathway of tryptophan degradation. It turns out... acid. That’s key because, unlike kynurenine, kynurenic acid can’t cross the blood-brain barrier. By lowering... lowering circulating levels of kynurenine in the body, the brain is protected from stress-induced biochemical...References: Skeletal muscle PGC-1α1 modulates kynurenine metabolism and mediates resilience to stress-...

  45. What's New in CRISPR - Spring 2019

    Type
    Blog Post
    Published
    March 21, 2019, 12:47 p.m.
    ...plasmids String assembly gRNA cloning (STAgR) String assembly gRNA cloning (STAgR), developed by Stefan...Read the paper in Genome Biology Find plasmids containing these base editors Spatially and temporally...gRNA multiplexing system that generates vectors containing high numbers of gRNAs in one overnight reaction... Read the paper in PLoS One Find the STAgR cloning plasmids Mobile-CRISPRi allows CRISPRi experiments...Cas12a inhibitors Using a bioinformatics and a screening approach, the Jennifer Doudna lab identified three...

  46. Quick, Versatile Plant Transgenesis with GreenGate Plasmids

    Type
    Blog Post
    Published
    Jan. 23, 2014, 3:23 p.m.
    ...introduced what’s been called a major breakthrough in cloning technology with the Golden Gate method. For the...researchers working in plant model systems. “The cloning is all done in vitro; it’s no different in mouse...to more tedious and time-consuming traditional cloning methods, Lohmann said. The key component of Golden... says their lab learned the hard way after the cloning method, which had been humming along at 99 percent... "GreenGate - A Novel, Versatile and Efficient Cloning System for Plant Transgenesis." PLoS ONE. 8(12)... Engler et al. "A One Pot, One Step, Precision Cloning Method with High Throughput Capability." PLoS ONE...

  47. March for Science

    Type
    Blog Post
    Published
    April 21, 2017, 2:30 p.m.
    ...however, inconvenient data must not be ignored. Returning to an earlier analogy, no amount of shouting will...scientists have chosen to leave the lab and march. Unintended consequences While frustration and anxiety have... science, others are concerned about possible unintended consequences of a march. Three of these risks...of everyone in government (19). To defend the meaning of the word ‘fact’. Policy must be based on our...science advocacy opportunities. Information for joining the march: March for Science: The website for...For science to be integrated into politics in meaningful ways, continued conversation about science’s ...: Universities Report Fourth Straight Year of Declining Federal R&D Funding in FY 2015 National Science...

  48. Multiple Plasmids at a Low Price: Kits from Addgene

    Type
    Blog Post
    Published
    Sept. 14, 2016, 2:30 p.m.
    ...useful kits you can order from the repository: FX Cloning Kit BIOFAB Plasmid Set pCri System Cell Free Protein...please write to us in the comments section.  FX cloning kit This kit is designed to be used with Type IIS...sites used in these vectors are of low abundance (meaning they are unlikely to be found in your gene of interest... incorporation of extensive sequences used for cloning into the final translated products. Negative selection... help decrease false positives observed in the cloning process, making it easier to produce the constructs...interest in a variety of model systems, thereby opening your research to a variety of experimental opportunities...fusions all use the same restriction enzymes for cloning. Furthermore, some plasmids contain peptides to...

  49. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    Published
    Dec. 6, 2022, 2:15 p.m.
    ...dynamics in live mice under a variety of behaviors, opening up new research directions that were inaccessible...concentrations in cells than existing sensors. Using genome mining and structure-guided engineering of potassium binding.... Figure 5: Structure-guided design and genome mining both led to new sensors with lower binding affinities...sensor plasmids here!   Torres Cabán CC, et al. Tuning the Sensitivity of Genetically Encoded Fluorescent... Indicators through Structure-Guided and Genome Mining Strategies. ACS Sens. 2022 May 27;7(5):1336-1346...cellular concentrations of lactate. The microfluidic screening platform developed here is also a promising approach...Figure 6: A) Schematic of BeadScan platform for screening candidate biosensor variants for performance under...

  50. Deep Dive: qPCR

    Type
    Blog Post
    Published
    Sept. 29, 2022, 1:15 p.m.
    ...appropriate choice (Panina, 2018). Look for studies specifically aimed at determining the best options for...Zhang M, Hu G, Fang F. Fudenine, a C-terminal truncated rat homologue of mouse prominin, is blood glucose-... amounts of reagents in your chemistry, unless running a multiplex reaction or on a rapid cycle machine... quick way to validate your primer probe is by running your qPCR product on an agarose gel, then cutting...real-time RT-PCR. J. Immunol. Methods 283:291–306.Panina, Y., Germond, A., Masui, S. et al. Validation of...Digital Droplet PCR for AAV QuantificationPlasmid Cloning by PCR...
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