...studies, where it could potentially label cells not directly connected to the injection site, or when using...volume. You’ll often hear that “high titer” is required for efficient AAV transduction, usually without...the optimal number of viral particles per volume required to express the gene of interest, in the cells ...are a few factors to consider. First, the titer required is inversely related to transduction efficacy:... recommended to optimize to the “lowest number required” instead of using the first amount that looks ...

...way to verify that the insert ended up in the direction you want - usually by Sanger sequencing or further...course there’s much more detail and verification required for the process to work well, so let’s go over... that the digest go until completion. The time required for complete digestion varies for different enzymes...associated manufacturers websites. These stains require you to either stain your gel after you run your...above, respectively). Some of the above stains require you to visualize your DNA using UV light – please...

... Material Transfer Agreement process that kits require; Nicole, the web master in charge of all our externally...You might find that your next paper is going to require help from a biochemist, a geneticist, a microscopist...world, so a process or project or mission that requires a broad knowledge base is going to necessarily...more things necessary to make a successful team. Entire books have been written about this! But this is...the type of team. Knowing what roles your team requires to function is certainly necessary, but equally...

... two very similar proteins. Additionally, they require variable expression of your target across samples...and it causes a large deletion that removes the entire exon that codes for the epitope recognized by your...expected target protein in a Western blot. You acquire a plasmid encoding your protein of interest, known...you very similar results, while the third looks entirely different (B), giving you confidence that your...amenable to high-throughput analyses, however, it requires access to a mass spec facility and the ability...

...Transposition protein: This is the protein that’s required for the transposon sequence to move from one DNA... transcriptase and integrase enzymes which are required for LTR retrotransposon transposition. Non-LTR...animals Transgenic animals are often generated by directly injecting DNA into the pronuclei of fertilized...dystrophin, the gene mutated in muscular dystrophy, require truncation to fit in viral vectors, but easily ... into eukaryotic and prokaryotic genomes. They require just two key components, the transposon’s DNA sequence...

...membrane, a threshold is reached and the neuron fires, sending the electrical signal down the axon (Lodish...subsequently photo-stimulated through fiberoptic cannulas directly implanted in the injection site and connected ... et al., 2008; Taylor et al., 2016; Hooper and Maguire, 2016). Recombinant adeno-associated viruses (rAAVs...Central PMCID: PMC4971574. Hooper, Andrew, and Jamie Maguire. "Characterization of a novel subtype of hippocampal...PubMed PMID: 4940442. Schnütgen, Frank, et al. "A directional strategy for monitoring Cre-mediated recombination...

...concentration of your reagent. Be aware that this approach requires excellent attention to detail, since you’ll end...Transfers are a little trickier than gels and may require multiple runs to optimize or troubleshoot.   Before...migrate…so don’t try eliminating one component entirely. If you are trying to transfer proteins of vastly...antibody.  Pro tip! If your antibodies are coming directly from tissue or supernatant, and you’re doing the... you don’t need to compare the protein amounts directly, stripping and reprobing. Just make sure to go...optimizing it. Blots for quantification generally require more optimization than blots used to confirm overexpression...

... a variety of Cas enzymes with different PAM requirements and editing abilities, such as base editing,...with multiplexed guides, mini screening libraries, direct edit optimization, and more. While there are many...modular nature of the Fragmid system allows for direct comparisons of CRISPR parts. The Fragmid system...fantastic way to save time and money on studies that require many assemblies or that compare the effects of ...construct. Once you’ve identified your experimental requirements, it’s a good idea to select an available kit...

...in a hyper-competitive environment where getting hired hinges on performing near miracles. When evidence...defending the role of science in our democracies will require constant vigilance and actions beyond marches, ...idea, but they might feel disconnected from the entire scientific process.  I've spent a lot of time debating...debating how to fix this.  As scientists, we work tirelessly in the lab where the groundbreaking research ...training as an academic scientist. I’ve worked tirelessly to get the publications, grant funding, and teaching...

... to optimize and troubleshoot the system. Site Directed Mutagenesis by PCR & Single Base Editing with ...Interestingly, this technology may make some uses of Site Directed Mutagenesis by PCR obsolete! Honorable mentions...give a great science talk from Addgene Executive Director Joanne Kamens. I highly recommend taking a look...August posts in chronological order here: Site Directed Mutagenesis by PCR Google Forums Round Up: First...

...the success of the company is attributed to the entire Addgene family; the depositors and requesting scientists..., Addgene supporters, and of course, Executive Director, Joanne Kamens, and founders, Melina Fan, Ken ...headed to the Museum of Science in Boston, where the entire Blue Wing was set aside for the event. As guests...family Perhaps the most heartfelt portion of the entire night was when Melina and Ken called their parents...

...community, Blugene will regularly be paired with a variety of science-inspired friends. As you can see on the... also made it hard for users to get where they desired. It became clear to us that we needed to improve... you can find what you need and feel happy and inspired to tackle your next science challenge. If you ...

...everyone involved. We’re adding self service to the repertoire of tools you can use to get support for our resources...our various FAQ categories or search through the entire FAQ collection. You’ll find topics ranging from...and press send. Doing so will bring your concern directly to our support team’s attention, where we can ...can then offer the same high level of direct service you’ve come to expect from us.   Try out the Help Center...

...resulting from past practices and mores.  His list is dire:  The current system is unsustainable, hypercompetition... tackle the issues with rules and practices of indirect costs.  The peer review and grant awards systems... encouragement.  Dr. Sibby Anderson-Thompkins, Director of the Office of Postdoctoral Affairs at UNC, ... put on good mentoring and applauded the new requirement for PIs to include in their annual NIH progress...

...disease” often requires understanding the disease genetics and the “treatment” usually requires drugs. While...validated for specificity to their target, or their direct target can be identified if it isn’t known. If ...undruggable’ proteins – having multiple hits or an entire pathway of possibilities greatly increases the ...

...time spent at the desk, but it is worth it. This requires a focus on deliverables. It does not mean a manager... and colleagues Sometimes this means you have to fire a good performer if they are enough of a jerk to... of most people, not just scientists) Are self-directed, but need precise leadership and support from ...Scientists. Subscribe to the Addgene Blog to follow the entire series as it is published. Good introductory reference...

...the recognition domains—including the CTD and the entire REC lobe—do not structurally resemble any other...weak, random binding to precision gene editing requires three major steps: RNA loading, DNA sequence recognition...The RNA guide sequence is extended as a single unpaired strand in the center of the protein. The repeat...1, nestled between the REC and CTD domains, is required for Cas9 nuclease function. Stem loops 2 and 3...But how to test DNA bases that are already snugly paired to each other? At this point, the protein can switch...

...this awesome experiment you want to do, but it requires some AAV. You’ve never worked with AAV before,...plasmids have a promoter, but this element isn’t required when the transfer plasmid harbors non-coding sequences...’s talk about some of the elements that aren’t required parts of an AAV transfer plasmid, but are often... control transgene expression. Lox sites are directional 34 bp sequences that can flank the transgene ...

...tagmentation preparation approaches described previously require a preliminary double stranding step. The double...single-stranded and both TA-based ligation and tagmentation require a double-stranded template.  Using viral genome...of serotype confirmation, this method does not require any additional reagents or lab work (Pacouret, ...polynucleotide chains. J Virol 9(2): 394-396. Lecomte E, Tournaire B, Cogné B, Dupont J-B, Lindenbaum P, Martin-Fontaine...

... my curiosity did not ultimately translate to a desire to do the research itself. Several years of exclusively...more creative faculties into motion, such that a desire to once again write for fun eventually saw an English...remarking to a classmate that, if this was one day required of me for work, I would have a high level of career...neurological health risk. Having played the sport my entire life, I was curious as to what the research had...

...wide variety of applications. But some techniques require you to select against the presence of a transgene... Antibiotic selection would only then kill the desired plants! PCR or fluorescent markers are currently...plants, but screening these can be challenging and requires specialized equipment. Reporters for studying ...from these plants will be green, containing the desired mutation but lacking both RUBY and the construct...

...and varying methods of replication. In fact, an entire classification scheme, known as the Baltimore replication...follow central dogma (gDNA to RNA to proteins) or require an intermediary DNA replication step between DNA...virus. RNA genomes can be transcribed into mRNA, directly translated into proteins, or, in the case of retroviruses...reproduce when experimentally necessary, genes for the required proteins are packaged in separate plasmids and...

...frequency. Although highly useful, this technique requires extensive sample processing to avoid disrupting...appear red. To enable this color switching, TRICK requires the following components: a GFP protein that ...field of view for constant tracking. You can then directly monitor multiple rounds of translation from this...caveats. In both cases, creating a reporter mRNA requires inserting multiple hairpin sequences into the ...

...researcher at the Wyss Institute for Biologically Inspired Engineering and member of the Addgene Advisory...production frustrating - a researcher can complete an entire lentiviral production protocol only to find that...widely requested and incredibly useful tools, they require extensive background work from the requesting scientist...Church lab at the Wyss Institute for Biologically Inspired Engineering and the Zhang and Doench/Root labs...

... favorite books - both scientific and not - to inspire you to read a little more this October. Science...science of race."     - Joanne Kamens, Executive Director  I Contain Multitudes by Ed Yong "Microbes are...another secret."     - Marcy Patrick, Associate Director of Quality Control Jonathan Strange & Mr. Norrell...Club (ABC) founder, Eric Perkins, is Addgene’s Director of Science Product Management and explains the...

...choosing a FP, but much of the primary data one might require when making such a decision remains spread across...to us for advice on choosing a protein, we often direct them there as a starting point.  However, there...previous versions.  Currently, all submissions also require a DOI referencing primary literature, and every...information on a particular FP; this can be done directly using the search bar on the homepage (and at the...

...(FP) require external light activation, you can’t use fluorescence to monitor processes directly affected...luminescent imaging: A luminescent substrate is required for imaging, and it is important to verify that...technology. Multicolor imaging using Nano-lanterns requires a linear unmixing algorithm to separate the various...

...blessed to have an incredibly supportive Board of Directors at Addgene. Our Board helps keep our focus on ...best practices and encouraging us to explore new directions. I would like to thank them for their time, dedication...can afford to do and when. Addgene’s Legal Team acquired permission from relevant plasmid depositors and...proud to be adding viral vector services to our repertoire. Additional Resources on the Addgene Blog ...

...membrane so DNA can be introduced directly into the cell. Microinjection requires the use of a fine needle to... bacterium’s genome through homologous or site directed recombination. Transduction is a common tool used...donor bacterium to a recipient bacterium through direct contact. The donor bacterium contains a DNA sequence...

...is not enough on its own to help you find your desired plasmid. In many cases, you will need a screening...you will need to sort through them to find the desired clone. Do your selection, then screen Why add a...what about identifying clones that contain your desired insert? This is where a  screen comes in. Let’s...process. This way, we can confirm the sequence of the entire plasmid. Please note that NGS verification is more...