...specific genomic locus: C-BERST Article contributed by Klaus Wanisch  Listen to the C-BERST podcast segment...processivity. Please find the MarathonRT plasmid here! Zhao C, et al. RNA. 2018 PubMed PMID:29109157. Nanobody ...Sontheimer lab used this technology, which they call C-BERST (dCas9–APEX2 biotinylation at genomic elements...in future studies of other genomic loci. Find the C-BERST plasmids here. Gao at al. Nature Methods (...protein fused with GFP, YFP, mCerulean, or other related fluorophores. Find all of the nanobody toolkit ...

...construct [1,2], i.e. positioning of the FP sequence relative to cell compartment targeting sequences, which...to characterize FP-fusion proteins functionally relative to the untagged protein of interest. To avoid ... retrieval sequence only functions at the extreme C-terminus [3]. You must therefore determine your cloning...strategy and decide whether you will create an N- or C-terminal fusion based on your experimental needs. ...

...response gene c-fos. For example, using Chr2 stimulation should result in robust increases in c-fos expression...stimulation compared to controls without Chr2. c-Fos is a reliable and widely used molecular marker of neuronal...proceed with your behavioral experiments. Comparison of c-fos amongst animals that receive different types of...neuronal activation, but it may still be possible to use c-fos expression to get the evidence you need. For example...activation in the novel context as determined by reduced c-fos expression, thus showing successful inhibition... as non-human primates [7, 8], zebrafish [9], and c. elegans [10]. If used properly, optogenetics is sure...literature to find all the applications of optogenetics relevant to your field. Ultimately, your question should...

...showed that iGluSnFR could detect neuronal activity in C. elegans, zebrafish, and mice. Second Generation ...Improved trafficking and localization through different C-terminal anchoring domains The original iGluSnFR in...adapted from Aggarwal et al… Figure 4. PDGFR is a C-terminal fusion to the PDGFR transmembrane domain ...mammalian expression pMinDisplay vector. GPI contains a C-terminal glycosylphostidylinositol anchor. SGZ contains...domain, followed by a modified form of the cytosolic C-terminal domain of Stargazin including a terminal ...give researchers the ability to monitor glutamate release from neurons and other brain cells in vivo. Glutamate...differentiating between synaptic vs. extrasynaptic glutamate release. Building on the previous generation of the SF-...

... standard strain, like DH5α, grown at 30 °C instead of 37 °C. If you’re unsure about the integrity of ...freshly harvested virus at 4 °C instead of storing it briefly at -80 °C. If troubleshooting, the safest...ultracentrifugation (75,000 - 225,000 g for 1.5 – 4 hours at 4 °C). After ultracentrifugation, virus may appear as a...freeze-thaw cycles on hepatitis B virus DNA and hepatitis C virus RNA quantification as measured with branched-DNA...restriction enzyme digest and compare it to some reliable controls. 2. Viral stocks may be sensitive to ...reducing the culture volume into which the virus is released, a more concentrated virus solution can be obtained...

...induce a lower inflammatory response within hosts. Relative to the other viruses discussed in this article...10.1016/j.gendis.2017.04.001 Maetzig T, Galla M, Baum C, Schambach A. Gammaretroviral vectors: biology, technology...2011 Jun;3(6):677-713. DOI: 10.3390/v3060677 Varanda C, Félix MDR, Campos MD, Materatski P. An Overview of...lentivirus and gamma-retrovirus are so closely related, their required components are very similar. While...

...can be very helpful if you're trying to find a relatively rare protein in an experiment with many proteins...short term storage (think days to weeks) in the 4°C. If you’ll only be using it every few month or if ...be going through small aliquots at a time, the -20°C is your best option, in aliquots no smaller than 10uL...

... present, it will repress expression of mKate2 relative to EBFP2. Weiss’s group used these reporters to...chain of BoNT/B into two fragments (BotNT-N and BotNT-C) and joined each fragment with one part of an established..., and showed that it can alter motor responses in C. elegans. Find the split BoTN/B plasmids at Addgene... it inactive, thus inhibiting neurotransmitter release. How is this light-mediated? The researchers split...visualizing the location and function of microbes rely heavily on fluorescent protein genes. These optical...

...J.G., Wohnoutka, P., Gerfen, C.R., Koch, C., Bernard, A., Dang, C., Jones, A.R., and Zeng, H. (2014). A ..., J. M., Vandenberghe, L. H., Haute, C. V., Thiry, I., Deroose, C. M., Mortelmans, L., Wilson, J.M., Debyser...overview see Addgene’s AAV guide. AAV particles are relatively small (20 nm) in size which makes them capable... Ravi, N., Wu, W.-L., Sánchez-Guardado, L., Lois, C., Mazmanian, S.K., Deverman, B.E., and Gradinaru, ...Winslow, B., Cain, N., Mihalas, S., Wang, Q., Lau, C., Kuan, L., Henry, A.M., Mortrud, M.T., Ouellette,... E., Tanaka, M., Kearney, M., Chattree, G., Yang, C. F., Shah, N.M., and Mooney, R. (2017). Identification...Res 406(1-2), 317-321. PubMed PMID: 2436717. Towne, C., Schneider, B.L., Kieran, D., Redmond, D.E.Jr., and...

...L-arabinose) without signs of toxicity or burden relative to a fluorescent protein control. We also added...visible in cells via TEM, and purified GVs under TEM. (c) Ultrasound imaging of tumor colonization by bARGSer-expressing...general method to create your own mARGAna cell line. (c) Z-projected phase contrast images of GV expressing...biomolecules. Appl. Phys. Lett. 110, 073704 (2017). Hurt, R. C. et al. Genomically mined acoustic reporter genes ...to produce GVs in their own lab using the most reliable method, which is the transient cotransfection ...

...could be a matter of timing, as jGCaMP7 was released relatively soon after GCaMP6. It could be a matter of...website.   Bibliography  Ersing, I., Rego, M., Wang, C., Zhang, Y., DeMaio, K. H., Tillgren, M., Fava, A.... E., Matho, K. S., Cetin, A., Woodard, K., Cepko, C., Guerin, K. I., Rego, M. A., Ersing, I., Bachle, ...10.3389/fnana.2019.00093 McDade, J. R., Waxmonsky, N. C., Swanson, L. E., & Fan, M. (2016). Practical Considerations...editor of Addgene’s eBooks — including the recently released Viral Vectors 101 2nd edition — I was incredibly...lessons learned from our plasmid lab to develop a reliable and robust high quality viral vector service, ...vector service because we realized that having reliable ready-to-use vectors made from plasmids in the...

...Gilmour, K. C., Adams, S., Thornhill, S. I., Parsley, K. L., Staal, F. J., Gale, R. E., Linch, D. C., Bayford...L., Quaye, M., Kinnon, C., Ancliff, P., Webb, D. K., Schmidt, M., von Kalle, C., Gaspar, H. B., Thrasher... PMCID: PMC7120651 Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander... Mansour, M. R., Schwarzwaelder, K., Bartholomae, C., Hubank, M., Kempski, H., Brugman, M. H., Pike-Overzet...PMC189872 Schröder, A. R., Shinn, P., Chen, H., Berry, C., Ecker, J. R., & Bushman, F. (2002). HIV-1 integration...integration for their life cycle to continue. They rely on a viral protein called reverse transcriptase ...lentiviral vectors. This is because most titering methods rely on isolating genomic DNA from transduced cells and...

...Paez-Espino, D., Ma, E., Witte, I. P., Cofsky, J. C., Kyrpides, N. C., Banfield, J. F., & Doudna, J. A. (2018)... L. G., Wolf, Y. I., Covarrubias, P. C., Shmakov, S. A., Silva, C., Tolstoy, I., Johnson, D. B., Koonin...and poorly understood. There are three subtypes, A–C, and they are grouped together because they contain...component of CRISPR — a nuclease effector. However, IV-C does in fact have a helicase domain due to a gene ...CRISPR as a technology. There are three subtypes, A–C, with the beloved SpCas9 (from Streptococcus pyogenes...–842. https://doi.org/10.1126/science.aav4294 Hu, C., Myers, M. T., Zhou, X., Hou, Z., Lozen, M. L., Nam... activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications. Molecular...

...pBR322 A Relaxed pColE1 ~15-20 ColE1 A Relaxed pR6K ~15-20 R6K* C Stringent pACYC ~10 p15A B Relaxed...pSC101 ~5 pSC101 C Stringent pBluescript ~300-500 ColE1 (derivative) and F1** A Relaxed pGEM ~300-500...held together with two hydrogen bonds not three as G-C pairs are. As a result, stretches of DNA that are ...number, ori, and incompatibility groups. Note the A -C compatibility grouping is an arbitrary designation...pMB1 (derivative) A Relaxed pBR322 ~15-20 pMB1 A Relaxed pET ~15-20 pBR322 A Relaxed pGEX ~15-20 pBR322...500 pUC and F1** A Relaxed pCDF ~20-40 CloDF13 (CDF) D Relaxed This table defines common cloning...replicate the host's chromosomal DNA, but they still rely on the host machinery to make additional copies....

...PCR or by evaluating visual phenotypes. Improving C. elegans fluorescent protein tagging with SapTrap ...called SapTrap to improve CRISPR genomic tagging in C. elegans. SapTrap offers scientists a convenient one...protein tagging systems For those who do not work with C. elegans, several groups have designed modular toolkits...by the Hornung Lab, allows users to easily create C-terminal tag fusions of endogenous genes in human ...plasmid containing the homology arms and an N- or C-terminal tag. The two PCR reactions are then mixed...line stably expressing Cas9. This PCR toolkit offers C- and N-terminal tagging vectors (eg GFP, Flag, YFP...the tag, selectable marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the...

...recent models of C. elegans molecular biology are based on the standard worm genome. While C. elegans are ...list of active spaces near you.   Learning about C. elegans with the OpenWorm Project One such space ...project. The main goal of OpenWorm is to simulate C. elegans as a computer model. When I first heard about...deconstruct and understand this complexity using existing C. elegans mutants. For instance, we use a mutant known...research goals in focus. Developing these skills relies upon biologists and computer scientists learning...neuron knockouts so that we can perturb factors related to worm motion. Working on the Cat Microbiome Project...

...common solution is to keep your plasmid at -20°C or even at -80°C, in this case your preparation can be eluted...periods of time, especially at room temperature or at 4°C. Storing DNA: temperature and longevity You should... stable for years. Plasmid DNA can be stable at 4°C or even room temperature for a short period, and there...hydrolysis of the glycosidic bond leads to the release of a purine (adenine or guanine) from the DNA. ...

...defined between a fairly nippy 20°C to a decidedly uncomfortable 45°C). Their more adventurous cousins ...of growth, say around 37°C, is a far cry from their native conditions of ~0°C, which drastically affects... But what about placing the mesophilic hosts at 0°C? That won’t work out either - most mesophilic systems... psychrophilic enzymes help them survive under severely cold conditions. To understand their unique importance...

...Park, J. C., Zhao, J., Luebbers, A., Green, E., Philibert, C. E., Zhang, H., Layne, M. D., Wu, J. C., & Garcia-Marcos...including "CLAP", combining an N-terminal CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed...human stem cell-derived neurons expressing ASAP5-Kv. C) Cultured rat hippocampal neuron expressing pan-membrane...the soma. The initiation site identified in panel (C) is indicated; red line is exponential fit. Adapted...the self-labeling tags SNAPf and CLIPf. These tags rely on synthetic dye molecules, which are much brighter...

...into the A2 noradrenergic area of the caudal NST. (A-C) Low-magnification images showing viral spread. (D-F...Backer, M. W. A., Brans, M. A. D., Luijendijk, M. C., Garner, K. M., & Adan, R. A. H. (2010). Optimization...Gruntman, A. M., Su, L., Su, Q., Gao, G., Mueller, C., & Flotte, T. R. (2015). Stability and compatibility...data free. Nature, 5–7. O’Connor, D. M., Lutomski, C., Jarrold, M. F., Boulis, N. M., & Donsante, A. (2019...Malaker, S. A., Poweleit, N., Maynard, L. H., Adams, C. M., Leib, R. D., Cirolia, G., Thomas, D., Stamnes...doi.org/10.1016/j.ymthe.2005.02.021 Zuk, P., Sanchez, C. E., Kostick, K., Torgerson, L., Muñoz, K. A., Hsu... AAV questions every year on a range of issues related to AAV vectors, many from people trying to answer...