... may be required for more complex experiments. A multiplexed experiment, for example, requires staining... negative controls Most biological experiments require positive and negative controls to ensure proper...is generally a sample or group that will have a desired response. For example, if you are studying the ...sample, the antibody binds specifically to its desired target (purple) and nonspecifically through the...The Preimmune serum control will not bind to the desired target but will bind nonspecifically through the... site.      Secondary antibody specificity  In indirect staining approaches, secondary antibody specificity...

...come up with a great experimental plan that requires indirect staining of both targets in your specimen.... are the same isotype and therefore cannot be indirectly probed simultaneously on the sample. What to ...matter? Isotype matters when constraining using the indirect method since a secondary antibody reactive to ...In the case above, you can’t costain with your desired antibodies because the pair belong to the same ...the same isotype can not be used for multiplexed indirect staining methods since the secondary antibody ...

...Direct reprogramming describes the process where differentiated cells are turned into a cell type of ...Though a valuable tool for regenerative medicine, direct reprogramming is an inefficient process, with the... the majority of cells failing to develop the desired identity. The development of single cell technologies...CellTag The Morris Lab developed CellTag to track the direct reprogramming of mouse embryonic fibroblasts (MEFs..."Single-cell mapping of lineage and identity in direct reprogramming." Nature 564.7735 (2018): 219. PubMed...pluripotent stems cells (iPSCS) and reprogramming and direct reprogramming Explore different methods for delivery...

...into primary cells, so most primary cell lines require viral infection. A new paper from Niels Geijsen...they could simply induce cells to take up protein directly? It’s not a new idea, but it comes with a few ...expression of a luciferase reporter. CPP was not required for protein entry into cells! After verifying ...non-detergent sulfobetaine 201 (NDSB-201) were required for protein delivery. D’Astolfo et al. coined ...take up, and continued protein expression would require multiple transduction events. For these reasons...concerns associated with virus work. Fourth, working directly with proteins removes the limitations of packaging...

...are required for the transduced cell to become light responsive than fluorescent proteins required to ...always be enough and therefore higher titer will be required. For example, retrograde AAV gets transported ...with your experimental setup. Some experiments require higher gene expression and therefore higher titer...if you end up concentrating your AAV above your desired titer and want to dilute it, err on the side of...less buffer than you would to achieve the exact desired concentration to avoid over-diluting, and make ...

...enabling genome-wide interrogation of the genes required for cellular processes. For a successful screen...plaques. ApoB bound lipoproteins (ApoB-LPs) are indirectly measured through their triglyceride or cholesterol...strong correlation with cardiovascular disease and indirect measurement of ApoB-LPs through triglyceride/cholesterol...et al, 2017).  In an effort to measure ApoB-LPs directly and develop a potentially high throughput in vivo...transcription. Transcription from σ54 promoters requires binding of bacterial enhancer binding proteins...difficulties such as small fluorescence changes or a requirement for very high excitation light intensities have...

...of the Nikon Imaging Center at UCSF. A common requirement for live cell imaging experiments is the ability...simultaneously. To do so with fluorescent protein labels requires multiple fluorescent proteins whose excitation... means that the choice of fluorescent proteins requires some thought. The first step in choosing fluorescent...fluorescent proteins can be separated from each other requires knowledge of their excitation and emission spectra...Beware that these infrared fluorescent proteins require biliverdin as a cofactor and so you may need to...Thorn is an associate professor at UCSF, where he directs the Nikon Imaging Center. He received his PhD in...

... interested in using GCE, here are the basic requirements of use. First, and most obviously, you need ... GCE are essentially those needed to make site-directed mutagenesis, many researchers new to GCE aren’...field, it’s not uncommon to find protocols that require reagents which aren’t broadly or easily available...complicated.” And Ryan Mehl would know. He’s the director of GCE4All, an NIH-funded center located at Oregon...phosphoserine and, because it uses ∆serB strains, does not require exogenously added phosphoserine. A community resource...using, GCE to reach out to them and let them know directly what protocols would be useful. Think GCE could...

...accessible by Cas9. Our previous blog post The PAM Requirement and Expanding CRISPR Beyond SpCas9 described ...a rationally engineered Cas9, SpCas9-NG, which required only the di-nucleotide PAM NG (Nishimasu et al...for base editing techniques, where Cas9 must be directed to a very specific site. Specificity vs. sensitivity...of different sites. Using a combined random and directed mutagenesis approach, the Anna Cereseto Lab created...specificity. Most recently, the Jungjoon Lee Lab used directed evolution to generate Sniper-Cas9 resulting in...Central PMCID: PMC5951633. Lee, Jungjoon K., et al. "Directed evolution of CRISPR-Cas9 to increase its specificity...

...maps have the identities of most features labeled directly on them making them much easier to quickly identify... see above) for your own reference or to paste directly into your notebook. This will make it easier for... immediate, high-level analysis, you can click directly on the “Analyze Sequence” button to pull up an...sizes, colors used to indicate them on the map, direction (if relevant), and types. Types listed are standard...sequences, binding site locations, lengths, and direction. Clicking on a particular primer here also highlights...Addgene software development teams for working tirelessly to implement these improvements. Let us know ...

...I like to envision that my research will have a direct impact on healthcare and the community. As a result...like-minded people, I quickly realize that a company requires more than just good data to become a product. ...settings, etc. What project in the Collins lab inspired Sample6? The initial work focused on using bacteriophages...graduate school usually) is one of the key skills required to achieve success in this business. A good presentation...expenditure of effort in preparation and practice is directly related to success presenting." A journey ...and succeeding. Take home Starting a company requires dedication, passion, and around-the-clock work...

...recognition sequence, they can be customized to direct assembly of DNA fragments. When designed correctly...contains sites with complementary overhangs that direct assembly of the final ligation product. As shown... be re-digested. In contrast, formation of the desired ligation product is irreversible because this construct...a single reaction! These overhangs specify the desired order of fragments, and the loss of enzyme recognition...Exonuclease-based methods like Gibson assembly require 20-40 bp of homology at the ends of DNA fragments...cloning is not 100% sequence-independent: to avoid undesired digestion, the Type IIS site used must not be ...

...other. In a direct approach, that is simple - but what about when you need to use an indirect (primary and... and to work in a variety of applications. The desired specificity of your secondary antibody depends ...expressed at very low levels or your application requires a high degree of affinity, then species choice...antibodies may be the better choice for assays that require high affinity binding, such as immunoprecipitations...multiple targets When staining multiple targets indirectly, you must strategically plan primary/secondary...

...distinguish proteins that directly interact with the bait from those that indirectly interact or from proteins...throughout a pulldown step, such as AP-MS. This requirement often prevents the identification of weak and...in a native cell contact. Other PPI methods may require protein interactions to remain stable (or to be...of Split-BioID is its speed of labeling. BioID requires 6-24 hours for tetracycline induced expression...available from Addgene! References Schopp, I.M., Ramirez, C.C., Debeljak, J., Kreibich, E., Skribbe, M.,...

...BritSciAssoc, @AAAS, @Stempra and science blogs like @WIRED Science. Don’t be scared to engage with them. Comment... careers are in advance of yours, but also get inspired and meet peers. Join societies like National Science...weeks at media outlets like National Public Radio, WIRED, Scientific American or National Geographic. Similarly...Citizen science projects Lastly, if you'd like to directly involve the public in your research projects, ...around climate change, biodiversity, or genomes requires lots of data. Get your community involved in sampling...up. My current job as a Marie Curie Fellow is a direct consequence of a blog that I started writing three...

...plasmid data directly into our file, one plasmid per row, as outlined by the column headers. Required columns...technology transfer office or other authorized office directly to make sure the deposit agreement/MTA is taken...be filled with short-answer text. You can also directly paste sequence data into the appropriate full ...and “low copy” if special growth conditions are required or if the plasmid is difficult to grow. Growth...Strain Other” field, and contact Addgene. We may require that you send your samples to Addgene pre-transformed...

...Washington Office Ben Corb - Director of Public Affairs at ASBMB Adam Fagen, PhD - Director, Early Career Scientist...perspective, while this is a move in the right direction, the increase notably still does not meet the ...governmental science funding and funding mechanisms require an engaged sector with effective advocacy skills...Scientist Segment at AAAS Marnie Gelbart, PhD - Director of Programs at Personal Genetics Education Project...their respective fields: David Cameron, PhD - Director of Media Communications, Harvard University Ray...

...Cas9. Cpf1 cleaves DNA in a staggered pattern and requires only one RNA rather than the two (tracrRNA and...cleavage pattern opens up the possibility of directional gene transfer, analogous to traditional restriction...which are difficult to modify through homology-directed repair. Both Cpf1 and its guide RNAs are smaller...mediate detectable off-target cleavage. Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target...that Cpf1 may improve the frequency of homology-directed repair (HDR) over non-homologous endjoining (NHEJ...sequence. Many research and clinical applications require HDR over NHEJ, so comparing HDR efficiency across...

...recover and the level of quality required. Some applications require very little DNA and you can get away... lower quality preps, while other applications require large amounts of high quality DNA. Figure 1 illustrates...quantity) – Bacterial transformation protocols require such small amounts of DNA (often on the picogram...the experiment, mammalian cell transfections can require quite large amounts of DNA (easily in the micrograms...spectrophotometry. Achieving the highest-quality DNA requires taking care at each step, but some purification... Using non-silica-based purification protocols requires less plastic and avoids the use of more hazardous...

...screening require X-gal, an expensive exogenously-added substrate, chromoproteins don’t require other substrates...detection. Unlike fluorescence or luminescence, which require UV lamps, fluorometers, or luminometers, chromoprotein...come from eukaryotes, they can be difficult to directly use in bacteria, where most genetic manipulations...recombination events: The plasmid carrying the desired genetic modification integrates into the genome...rhamnose-inducible promoter. The resolution step leads to the desired mutation and excision of the vector backbone. To...

...consider when time is of the essence. This method requires a vector backbone containing a topoisomerase I...particularly tight? Most cloning processes will require a PCR reaction or two to generate templates for...to look at the prices of the additional enzymes required. FastCloning may be the least expensive of all...all the options discussed as it only requires PCR and the inexpensive DpnI enzyme. Otherwise you let your...restriction enzymes. In Golden Gate cloning, the desired end construct does not contain the type IIS recognition...This feature biases the reaction in favor of the desired assembly to make the process close to 100% efficient...

... for which the underlying genetic cause is not entirely known. Identifying which genes are important for...experiments investigating the role of individual genes or entire pathways in a particular disease process. While...double-strand break in the target DNA, which, when repaired through error-prone non-homologous end joining...libraries after amplification and after a screen requires the use of next-generation sequencing. Representation...sufficient representation within your mutant population requires a massive amount of cells as starting material... be independently validated to ensure that the desired modification produces the phenotype you screened...

...virulent viruses (it attacks the immune system directly). It may seem odder when you consider that out...derived from the host/producer cell membrane, and acquired as an envelope during exocytotic budding, as the...first steps! However, retroviral vectors can still direct the translation of proteins because they integrate...phrase — “pseudotransduction” — to describe the direct protein transfer of RT, which would actually occur..., 1971) and the full wild-type viral genome can direct the production, packaging, and release of progeny...., Goodman, H. M., & Bishop, J. M. (1973). RNA-directed DNA polymerase of Rous sarcoma virus: Initiation...

...to transformation (Figure 1). As this approach requires no special reagents or purification kits, it is...primary method to produce the linear fragments, as desired modifications and homologous regions can be encoded...fine, yet performing multiple modifications will require ultracompetent (109 CFU/μg) cells. Primer design... is discussed in more detail below. The main requirement for IVA cloning is carefully designed primers...order primers that bind at either side of the undesired portion, designed to amplify the rest of the vector... (Figure 3). Achieving multiple modifications requires multiple recombination events to assemble the ...

...were fused in-frame to a tethering partner, which directed localization of the fluorophore, and thus enabled...529nm human α-Tubulin Tubulin Repeatable, directional cloning   The principle behind MXS-chaining ...MXS-chaining also has the advantage of being directional, meaning that (in the previous example) one can.... For example,  chaining-based cloning methods require that the separate modules combined using these ... of the modules, because neighboring fragments require compatible cohesive ends Engler et al., 2008 ...Methods Method Restriction Enzymes Chaining Direction MXS-Chaining SalI and XhoI MluI BioBricks ...

...Addgene is named Sonic Hedgehog but this is not a direct reference to the video game character but rather... sqh.   cookie monster, a meiotic arrest gene required for spermatogenesis One of our smaller conference...Jiang and White-Cooper, 2003). Why call a gene required for spermatogenesis after a Sesame Street character...: 8101173.   Bodmer, Rolf. "The gene tinman is required for specification of the heart and visceral muscles...light chain–activating phosphorylation sites are required for oogenesis in Drosophila." The Journal of cell... myosin is encoded by spaghetti-squash, a gene required for cytokinesis in Drosophila." Cell 65.7 (1991...

...complex datasets into the desired repository. They also support entry of required metadata, with the option...reproducibility RSpace was designed in response to requirements that emerged from intensive engagement with ... ELN is only ever a small fraction of all data directly relevant to a particular experiment, series of...the ELN, in some cases the integrations provide a direct boost to reproducibility. For example, it’s possible...has to make it easy to export data from the ELN directly into at least one and preferably more than one...

...found in the same gene. If we only have small, directed editing abilities, any gene editing treatment ...dragging and dropping in a functional copy of the entire gene, allowing a one-size-fits-all approach to ...PASTEsystem involves insertion of landing sites via Cas9-directed reverse transcriptases, followed bylanding site... recognition and integration of cargo via Cas9-directed integrases.   Use PASTE can be delivered via...large insert techniques like HITI or HDR, which require specific repair pathways to work, PASTE is more...genome insertion without DNA cleavage with CRISPR-directed integrases bioRxiv 2021. 11.01.466786; doi: https...

... Cpf1, but they require both a tracrRNA and crRNA to cleave target DNA (Cpf1 requires only a crRNA for...Cas13a (C2c2) 2x HEPN crRNA RNA 3' A, U, or C (not required by all orthologs) Specific RNA cleavage (followed... PFS. This is in stark contrast to Cas9, which requires that each DNA target have high sequence identity...to limit the spread of infection throughout the entire population. This property of Cas13a opens up the... from 20-28 nucleotides in length and does not require a specific PFS, making it a very flexible cleavage...

...researchers at the Wyss Institute for Biologically Inspired Engineering. CRISPR/Cas9 is an enormously plastic...pass. In addition, activation experiments often require quite a bit of tuning before you know your system...should also be ready to test three or four guides directed towards that gene as there can be a large difference...rather than going through all the troubleshooting required for Cas9-based activation. CRISPRa for genome-...providing a new gRNA rather than engineering an entirely new protein. This makes it much cheaper to use...researcher at the Wyss Institute for Biologically Inspired Engineering.     Alejandro (Alex) Chavez is a ...