...but you should ensure that the bases do not result in the formation of a hairpin structure within your primer...You should select an annealing temperature based on the melting temperature (Tm) of the portion of the primer...The process is shown graphically in the following figure, in which we add EcoRI and NotI sites to Your Gene...you can use a pretty wide range of annealing temperatures, but you may need to increase your primer length...

...the QCRG to continue our work toward a cure for COVID-19, be sure to follow us on Twitter (QBI_UCSF and...do anything and everything we can to help find a cure -- and quickly. Normally drug discovery is a process...much faster. Years of science, done in weeks Figure 1: Sending out 80 FedEx envelopes in a single day...want to be careful with these clones (and help us figure out what is going on there!).  Find the Krogan ... be working on this problem more quickly. Figure 2: Getting our research in line, with a row of ...

...ApoB bound lipoproteins (ApoB-LPs) are indirectly measured through their triglyceride or cholesterol contents...correlation with cardiovascular disease and indirect measurement of ApoB-LPs through triglyceride/cholesterol ...al, 2012; Lawler et al, 2017).  In an effort to measure ApoB-LPs directly and develop a potentially high...Athersclerosis 2012. Pubmed PMID: 23068583 Thierer et al, Nature Communications 2019. Pubmed PMID: 31366908 Adapting...CRISPR activation plasmids at Addgene. Liu et al., Nature Communications, 2019. PubMed PMID: 31451697. Genetically... and offers many advantages over traditional measurements such as patch clamp recordings. However, technical...

... of being a “good scientific citizen” and making sure you do what you can to help your fellow researchers...subtle, and it can take a lot of detective work to figure them out. But, when they come to light, it’s clear...way so other researchers can use them, to making sure that methods and protocols are available and detailed...Press we developed STAR Methods to improve the structure and transparency of methods sections, including... room to do more. If we could really instill a culture of rewarding good scientific citizenship in all...making it happen would increase dramatically. I’m not sure what the best approach to doing that will be, and...

...and the guides are relatively unstable.   Figure 1: CRISPRainbow labeling. Two MS2, PP7, or boxB...interest, the team could visualize three loci and measure inter-locus distances. They tested this system ...intergenic DNA region (IDR3) as a reference loci to measure inter-locus distances of loci pairs from intergenic...variation (CNV) analysis of the U2OS cells.   Figure 2: CRISPR-Sirius tricolor labeling. (A) Diagram...dCas9 and engineered sgRNAs using CRISPRainbow.” Nature Biotechnology 34 (2016): 528-530. PubMed PMID: ...scaffolds for signal amplification in genome imaging.” Nature Methods 15 (2018): 928-931. PubMed PMID: 30377374...

...are secreted from the cellular membrane into the culture medium. AAV particles are either associated with...vexosomes (vector-exosomes), exosome AAVs, or exo-AAVs (Figure 1). The researchers discovered that these particles...differential centrifugation steps to separate cell culture supernatant containing exo-AVVs from producer cells...then to enrich the microvesicles containing AAVs (Figure 2). Since the development of the exo-AAV isolation...the production of exosomes or stabilize their structure or their interaction with AAVs (Schiller et al...model of glioblastoma (Volak et al. 2018). The structure of exosome AAVs is related to the use of microvesicles...

...and cytoplasmic (CKB1) RNA target. Adapted from Figure 3 Colognori et al 2022.   Find CRISPR Csm plasmids...lagged. Fixation, chemical staining, and light exposure can negatively affect FPs during these processes... also withstands denaturing conditions, high temperatures, and other chaotropic conditions better than...these preparations. After crystalizing the hfYFP structure, the group rationally designed GFP variants with...fluorescence in high molarity GdnHCl, at high temperatures, and in the presence of oxidizing agents. Adapted...Adapted from Fig. 2 Campbell et al, Nature Methods   Find YFP plasmids here!   Campbell, B.C., et ...

...cleave them leaving mature crRNAs bound to the Cas9/tracrRNA complex. D) The mature crRNA is used to guide...GAA, or GAT SpCas9-NG 3' NG  Staphylococcus aureus (SA); SaCas9 3' NNGRRT or NNGRR(N)  Acidaminococcus...the crRNA to Cas9 and facilitates processing of mature crRNAs from pre-crRNAs derived from CRISPR arrays...unique stretches of DNA between them “spacers” (see figure below). After years of research, we now know that...sequences required for processing pre-crRNA into mature crRNA and tracrRNA binding. The spacer regions,...

...phosphorylation, promoting transcription (see figure below). Subsequent exposure to red light rapidly deactivates ...limited studies and can fluctuate with changing culture conditions. The use of low intensity light solves...promoting transcription of a GFP reporter. Subsequent exposure to red light rapidly deactivates the system. Version...systems The large dynamic range of these systems ensures that they can be used with many types of proteins...Scouras A, Ellington AD, Marcotte EM, Voigt CA. Nature. 2005 Nov 24;438(7067):441-2. PubMed. Find the...

...important considerations for the future of open science? What about the future of open science are you most...do you hope Addgene will accomplish in the near future? Katz: I’m excited that Addgene is continuing to...those “what could go wrong scenarios” and making sure we have adequate process and controls in place to... particular area or profession, you need to make sure you have learned and mastered the fundamentals.   ...was a founding member of Mass Medical Angels, a venture mentor at MIT and served on the Boards of BioBuilder...

...cell line cultures. Lentivirus Lentivirus is a type of retrovirus and shares many common features with gamma-retrovirus... gamma-retrovirus including genome architecture, host integration, and its association with disease. The...cell types. Altogether, AAV has many excellent features for human gene therapy and is currently being ...types of interest. This is a particularly nice feature as organ-specific injections, or the location of...   Table 1: Types of viruses and their features Choosing a virus Now that we’ve reviewed the...

...provided by the manufacturers and enables customized experiments. Projects such as the OpenFlexure Microscope...modify the device for individual experiments. Manufacturers rarely provide the construction plans or CAD...accessible open-source hardware. Projects such as the OpenFlexure microscope or open-source modular optical toolbox...documentation, which facilitates the reproduction of the structure in a similar way to the biological protocol, and...et. al, "Robotic microscopy for everyone: the OpenFlexure microscope," Biomed. Opt. Express 11, 2447-2460...

...densities are captured at the 17%, 25%, and 60% layers, and genome-containing virions are captured at the 40%...pun intended.)  Steps to clear writing Let’s use Figure 1 as our example. Remember, our goal is to use ...same information as sighted readers.    Figure 1: An image from AAV Purification by Iodixanol ...What are they hoping to get from it? The image in Figure 1 appears on our protocol page for AAV Purification...group to share with the rest of the workshop.  For Figure 1, Addgene chose this alt text: "The left panel...

...focus on antibodies that recognize proteins. Figure 1: Steps in the generation of hybridomas for monoclonal...fused to myeloma cells at a 1:1 ratio and then cultured in a special medium called HAT. This medium selects...antibodies are particularly useful when you want to ensure that the antibody binds to a specific region of... during your experiment, then you’d want to make sure that you choose an antibody that recognizes an epitope...polyclonal antibodies - which are a heterogenous mixture of antibodies that recognize many different epitopes...plasmid to create a recombinant antibody.     Figure 2: Changes to the heavy chain or light chain genes...

...negative (digital) signal at the end of the reaction (Figure 1). Partitions that did not contain any target ...precision. This means digital PCR provides an absolute measure of target abundance, does not require standard ...directly at the predicted double strand break site (Figure 2A). The single molecule resolution of digital...completely “blind” to this very important difference (Figure 3). If you are interested in harnessing the power...requires target DNA sequencing to confirm the exact nature of your mutations. A great description of the plethora...induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology 31, 822–826 (2013). PubMed PMID: ...

...scientists and nonscientists alike). My only in-depth exposure to neuroscience prior to this conference was a.... This was not at all the case. The conference featured people who work in sociology, behavior, and artificial... session organized by Story Collider and which featured a talk about the different parts of the brain ...getting into neuroscience from the mol bio side you’re sure to have many opportunities to learn about and develop...website and on the Addgene blog to get some ideas for future experiments and to find tools that will make your...Ed Boyden on Optogenetics, Neuroscience, and the Future of Neuroengineering Primer on Optogenetics Resources...

...powerful approach for proteomic mapping in both cell culture and whole organisms (see our 2019 overview). Until...doi.org/10.7554/eLife.64631  Goertsen D, et al. Nature Neuroscience. 2022. https://doi.org/10.1038/s41593... applies both positive and negative selective pressures for novel capsids. In a continuation of that work...Find M-CREATE plasmids here!  Goertsen D, et al. Nature Neuroscience. 2022. https://doi.org/10.1038/s41593... factors, including limitations of number of flourescent proteins that can be inserted into a cell without...visual barcode plasmids here! Kaufman, et al. 2022. Nature Comm.https://doi.org/10.1038/s41467-022-30008-0...

...understanding the brain’s function is knowing its architecture, in particular the connections between different... uptake of these dyes in vivo, microinjection, pressure injection or iontophoresis (applying small electric...of RABV. This improvement resulted in a clearer picture of neuronal connectivity in the mammalian brain...but overall with rather low efficiency. Not all measures of transport efficiency are consistent which may...observations like these may be a starting point for future developments in the field of AAV vectors. Conclusion...achieve transgene delivery by targeting peripheral structures (such as injection into muscle) as opposed to...(2008). Concepts in the pathogenesis of rabies. Future Virol 3(5), 481–490. PubMed PMID: 19578477. PubMed...

...and work quickly to mitigate RNA degradation. Make sure to use DEPC-treated or RNAse free water. If using...water, autoclave the water to inactivate the DEPC.  Ensure that plasticware or glassware used is RNase free...or more large, expensive ultracentrifuge.  Figure 1: An outline of the different steps in RNA extraction...you need really clean RNA, it’s important to make sure that when extracting, to only take the aqueous layer... from) on your resuspended RNA sample will help ensure DNA contamination is not an issue.  References...thiocyanate–phenol–chloroform extraction: twenty-something years on. Nature Protocols 1:581–585. https://doi.org/10.1038/nprot...

...the biggest project I’d seen in my already long tenure at Addgene. I would work with our research team...of what we discussed and decided. I needed to a) figure out the sources of miscommunication and b) find... steps towards solving this problem by 1) making sure we qualified these words when we discussed them ...Creating this guide was relatively easy. I took a picture of virtually every container we used, then for ...team identify possible points of confusion in the future. Improving cross team communication, be it in ...productive. Higher Productivity. Taking active measures in improving cross team communication has helped...