... 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...

...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...

...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...

...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 ...

... 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...

...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...

...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 ...

... 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...