...these topics. Listen to Part 1 Here Listen to Part 2 here! Time : Topic 0:00 - 0:50 : Intro 0:51 - 3:08...Improvements that can be made to the grad school process 22:41 - END : Niroshi and Ben's favorite advice...Future of Research Looks to Improve the Research Process  Browse our Career Posts Resources on Addgene....

...commonly used in optogenetics: 1) electrophysiology and 2) behavioral experiments. I’m not an electrophysiologist...choice in the brain region you wish to stimulate 2) Fabrication and implantation of the optical fiber...thoroughly elsewhere (here and here for instance) [2], [3]. Karl Deisseroth of Stanford University , one...reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn about the...monitor expression at various time points (e.g. every 1-2 weeks over 8 weeks) in order to determine the optimal...(4):251-66. doi: 10.1038/nrn3171. PMID: 22430017. 2. Fenno L, et al. The development and application of...analysis of microbial opsins. Nature methods. 2012;9(2):159-72. doi: 10.1038/nmeth.1808. PMID: 22179551; ...

...testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two different ...selection process. Factors to think about are (1) your experimental conditions and measurements; (2) the cells... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLOS ONE, 8(9),... Comparison of four promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1...analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral...will allow you to efficiently move through this process.  Many other factors can also affect your AAV experiments...

...Nature Cell Biology, 26(2), 294–304. https://doi.org/10.1038/s41556-023-01342-2 Koblan, L. W., Arbab, M...order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent...region of separate strands called an R-loop (Figure 2). This allows the guide RNA to base pair with the ...accessible on the outside of the enzyme. Figure 2: CRISPR R-loop. Created with BioRender.com. Base...), 35–40. https://doi.org/10.1038/s41587-020-0592-2 Additional Resources on the Addgene blog CRISPR 101...inhibitor of base excision repair (BER), the cellular process that catches mutated U’s in DNA and ideally converts... In this case, the team suspected that the BER process was excising the incorrect U base, creating an ...

...developed around how they replicate (Figure 2). Figure 2: Baltimore replication classes. Image credit...safer viral vectors that can be used in BSL-1 or BSL-2 labs. Viral vectors are viruses that have been genetically...and eukaryotes, which use a binary replication process whereby one cell copies itself and “splits” into...” genome with genes central to the replication process removed. This means the vector can deliver a genetic...

... analogs such as 2-fluoroadenine (2-FA), organisms with functional APT will convert 2-FA to cytotoxic ...selection of plants undergoing RNAi, and LIIbeta F 1-2 RNAi, which allows assembly of intron-spliced hpRNAs...nucleotides, resulting in death. In the presence of 2-FA, potent gene silencing of APT is required for survival...block of our understanding of complex biological processes. Many tools exist to perturb biological function... interference (RNAi) is a conserved eukaryotic process where approximately 20-30 nucleotides of double-stranded...formation of a hairpin RNA structure, resulting in processing by the endogenous RNAi machinery. Alternatively... engineered to created artificial miRNAs. The processed miRNA or siRNAs then silence the target gene(s...

...Currently available from the Kim lab are 2 presynaptic and 2 postsynaptic targeting mGRASP plasmids. Additionally... cells were pulsed with 2µg/ml of Doxycycline for 2 hours, and imaged on the Keyence BZ-9000 fluorescence...work! Robinson et al., Dev Cell 2010 Feb 16;18(2):324-31. Cheeseman et al., J Cell Sci 2013 May 1...promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated... of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly...1 modules can be similarly assembled into a Level 2 vector, thus forming a functional genetic circuit....circuit. Flexibility has been built into the Level 2 vectors to allow for additional iterations of Level 1 assembly...

...satisfactorily accepts DNA as its substrate (see figure 2).          The artificially evolved adenosine deaminase...editing platforms for plant systems.  Figure 2: Plant base editor platforms and their molecular components...available for plant systems. Target-AID based (CBE-2)-  and rAPOBEC based (CBE-1)-  CBEs are successful...based CBEs are known to deaminate cytosine placed in 2-6 bp and 4-8 bp, respectively, of the protospacer....PMID: 27096365. PubMed Central PMCID: PMC4873371.  2. Nishida, Keiji, et al. "Targeted nucleotide editing...capability with the endogenous tRNA-processing system." Proceedings of the National Academy of Sciences...

...Cas9 from Streptococcus pyogenes.   Figure 2:  Crystal structure of S. pyogenes Cas9 in the apo...NUC and REC lobes laying open like a book (Figure 2). The portion of the CTD that recognizes the PAM is...it doesn’t appear in the crystal structure (Figure 2) at all. When this crucial recognition region is disordered...regions, so crystal structures like those in Figures 2 and 3 don’t show these regions, even if they are present...itself at the bottom of the image. Stem loops 1 and 2 extend horizontally across both lobes. The guide RNA...is required for Cas9 nuclease function. Stem loops 2 and 3 are not strictly necessary for in vitro cleavage...open conformation, but highly flexible, as in Figure 2. The guide RNA is bound primarily by the REC lobe,...

...optimal sybody candidates. Figure 2: Outline of the process for selection of sybodies against membrane...identify and isolate sybodies targeting the SARS-CoV-2 Receptor Binding Domain found on the membrane-bound...Generation Toolbox kit. Find the Seeger lab's SARS-CoV-2 sybodies! References Geertsma ER (2013) FX Cloning...2020) Synthetic nanobodies targeting the SARS-CoV-2 receptor-binding domain. BioRxiv.  https://doi.org...sybody), ribosome, and mRNA. During the screening process, displayed sybodies that bind to the biotinylated...

...possible when each of the XFP genes is introduced at ~2 copies per cell per color, following a Poisson distribution...(prepared with Addgene #104110 - #104112) (Figure 2). We’ve also created constructs with containing chemical...AAV titer - The XFP genes have to be introduced at ~2 copies per cell per color to achieve the highest color...samples and visualize. For more details on this procedure, find the protocol here.   Tips and troubleshooting...Ultrafast tissue staining with chemical tags." Proceedings of the National Academy of Sciences111.36 (2014...

...mechanism of action are presented in Figure 2. Figure 2: Single-stranded donor DNA interaction with...Nature biotechnology (2016). PubMed PMID: 26789497.  2. Lin, Steven, et al. "Enhanced homology-directed human... a biological process in detail often suggests new strategies to manipulate that process. We began our...

...agarose. Proceedings of the National Academy of Sciences of the United States of America, 76 2, 615-9 ....alkaline lysis plasmid miniprep Starting material: 2 mL bacterial culture Product: plasmid DNA This kit-free...protocol: 1) it solubilizes both DNA and agarose, and 2) it helps DNA stick to the glass. After binding DNA... had a similar level of sensitivity. Figure 2: Steps to purify DNA using a cellulose dipstick. Image...and without degradation during the extraction procedure. Additionally, purified DNA was free of agarose...

...is excised from the donor vector.   Figure 2: The Gateway system adopts phage integration into ...our website to search for your favorite gene! STEP 2: Generate an Expression Clone When making the expression...expression construct in only 1 day, as opposed to 2+ days with traditional restriction and ligation cloning...Clon Transgen 4:138. doi:10.4172/2168-9849.1000138 2. Hartley JL. Use of the Gateway System for Protein...using Gateway technology To better understand the process, we’ll walk through an example experiment where...directly into destination vectors. The cloning process is simple -  no restriction, ligation or gel purification...

... a population in a procedure termed fluorescence activated cell sorting or FACS (2). By analyzing single...expression. A routine flow cytometry experiment uses 2 or 3 different fluorescent colors each measuring a...distinct populations on a FSC versus SSC plot (Figure 2).  A user can then “gate” around the specific population...cell and T cell into distinct populations (Figure 2). An immunologist who studies B cells, might then ... B cells, plasma cells or others.   Figure 2: Due to differences in their size and complexity, ...forces the cells to line up in single file in a process termed hydrodynamic focusing. This allows each ...factors including sample preparation, staining procedures, and controls. To prevent instrument clogs, ensure...

... working with is correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...colonies (5-10) and grow them in a small culture volume (2 or 3 ml) containing the corresponding antibiotic, ...scientist at Addgene. Cloning can be quite an arduous process. The PCR could fail to produce a product, the transformation...can go wrong! With all the steps in the cloning process, there are also many ways to troubleshoot the cloning...Addgene. In the lab he develops new assays and processes. Outside the lab, he enjoy the outdoor in all ...

...conditional knock-in cassette. As shown in Figure 2, the cassette should contain a reporter DNA sequence...a targeted neuronal population.    Figure 2 (modified from Schnutgen et al. Nat Biotech 2003):...cellular level in the mouse. 2003 PubMed PMID: 12665802. 2. Branda, CS and Dymecki, SM. Talking about a Revolution... SM. 2008. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range ...SSR of choice is introduced, recombination can proceed either by first utilizing the loxP sites or the...

...guide expression and dsDNA breaks. Figure 2: Non-targeting vs. safe-targeting guides. The genome-wide...target all protein-coding genes present in the genome; 2) non-targeting guides (red), which target no sequence...guides that had zero mismatch off-targets. Guides with 2-bp mismatch off-targets were only toxic if they had...tolerated than mismatches more distal to the PAM, and 2) guides with high GC content have greater off-target...PMID: 28474669. Pubmed Central PMCID: PMC5424143. 2. Tsai, Shengdar Q., et al. "GUIDE-seq enables genome-wide...for identifying genes involved in a biological process. These screens provide an alternative to genome-wide...

...require application submission in-house approximately 2 weeks before the RFA due date in order for the administrative...deliverables to the aims requested in the call. Step 2: Develop a research plan Most grant applications ...proposal typically lists the deliverables, explains 1-2 potential pitfalls of the intended protocol, and offers...want to mentor you during the grant application process. When my PhD advisor first asked me to write a ...PI asks for assistance in the grant application process, she has faith in you! Additionally, keep in mind...motivating research will help you understand the process and continue to improve your grant writing skills...Still want more information on the application process? What types of writing feedback do you find most...

... fluorophore excitation by absorption of a photon 2. Energy transfer from the excited donor directly to...PMID: 24194740. PubMed Central PMCID: PMC3810607. 2. Bajar, Bryce T., et al. "A Guide to Fluorescent Protein...PubMed PMID: 10500161. PubMed Central PMCID: PMC18018. 2. Miyawaki, Atsushi, et al. "Fluorescent indicators...orientation of the FRET pair. Since many biological processes occur within the typical FRET range, FRET efficiency...fluorophore on the same protein and are useful when a process affects the conformation of the probe. More on ...answer sophisticated questions about cellular processes. Currently, FRET experiments can probe protein-protein... activity of small molecules, detect cellular processes and signaling cascades, quantify mechanical tension...