...Cas9 + Empty sgRNA) 47549 C. elegans yes, cut S. pyogenes Goldstein DR274 42250 C. elegans BsaI none S. pyogenes...gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish Xenopus CRISPR Resources ...pyogenes Zhang PX854 62886 Mammalian BbsI yes, cut, C-terminal S. pyogenes Zhang pGuide 64711 Mammalian ...pyogenes Zhang PX852 62884 Mammalian BbsI yes, cut, C-terminal S. pyogenes Zhang PX855 62887 Mammalian BbsI...pyogenes Zhang PX856 62888 Mammalian BbsI yes, activate, C-terminal S. pyogenes Zhang pU6-(BbsI)_CBh-Cas9-T2A-BFP-P2A-Ad4E1B...Bullock and Port pCFD2-dU6:2gRNA 49409 Fly BbsI none S. pyogenes Virmilion Bullock and Port pDCC6 59985...Drosophila BbsI none S. pyogenes Virmilion Bullock and Port pAc-sgRNA-Cas9 49330 Drosophila BspQI yes, cut S...

...Analyze a Restriction Digest References 1. Henrici, R. C., Pecen, T. J., Johnston, J. L., & Tan, S. (2017)....Generating DNA Molecular Weight Markers. Scientific Reports,7(1). doi:10.1038/s41598-017-02693-1. PubMed PMID...

...References and resources References Chadeuf G, Ciron C, Moullier P, Salvetti A (2005) Evidence for Encapsidation...Lindenbaum P, Martin-Fontaine M, Broucque F, Robin C, Hebben M, Merten O-W, Blouin V, François A, Redon...production process? These questions are especially important when producing multiple vectors in parallel because...

...temperatures (up to 95 °C) and is active over a range of elevated temperatures (37–75 °C). T4 DNA ligase ...PMC4737175. 22. Liu, Pingfang, Artur Burdzy, and Lawrence C. Sowers. "DNA ligases ensure fidelity by interrogating...commercially available (see below). Conclusion It is important to note that thermostable, high fidelity, nick-selective...

...Discovers America. New York./  Callahan, K., Peterson, C., et al. 2022. External collaboration results in student...Evolution and Development. 24(5):127-130. Kimball, C., Powers, K., Dustin, J., Poirier, V., Pellettieri... outcomes and the opportunity to make new discoveries. Provost notes the importance of acknowledging student...history. Interestingly, he also adds to the disproportionately large number of honorees to have trained ... and he wanted to provide the same kinds of opportunities for his students. But he also found that his...Bringing his research into the classroom. I had the opportunity to talk with Provost recently about his development...Undergraduate Research Experience (CURE). Having incorporated a discovery-based lab on planarian regeneration...

...conformation capture (3C) based methods, such as 5C or Hi-C (Han et al., 2018). Think of 3C methods as taking ...other 3D chromatin methods such as ChIA-PET or Hi-C, by increasing both the number of unique chromatin...but enhancers and chromatin looping play important supporting roles. Enhancers are cis regulatory DNA ...networks’ of 157 super enhancers.  3. Measuring temporal changes in chromatin interactions A third application...application for CAPTURE 2.0 is detection of temporal changes in chromatin conformation, like those that ...interactions. The chromatin story told by CAPTURE 2.0 is important, but it’s not the only plot line in gene regulation...Multiplexed capture of spatial configuration and temporal dynamics of locus-specific 3D chromatin by biotinylated...

... epitope such as c-Myc, GFP or V5 can be used to tag the protein of interest on the C- or N-terminus. ...Their porous surface provides a large area for binding, but antibodies conjugated within the pores are ...used for IP. These proteins bind only to the Fc portion of the immunoglobulin, leaving the antigen binding...or a magnet (magnetic beads), providing a solid support while unbound proteins are washed away.  While ...to be adjusted depending on the sample. It is important to remember that sample lysates will also contain...

...predictable kinetics over 14 hours of expression in C. elegans embryos. However, this initial timer was ...timer could be used in vitro, in mammalian cells, in C. elegans, and in Xenopus embryos. Of course, timers...have been around since 2000 when Terskikh et al. reported the production of dsRed E5. This timer predictably... your experimental setup less cumbersome. 3. Portability across multiple systems Terskikh et al. showed...fluorescent timers that change color from blue to red report on cellular trafficking." Nature chemical biology...Chronology of islet differentiation revealed by temporal cell labeling." Diabetes 58.8 (2009): 1863-1868...

...calculate the viral titer: GC/mL = {[(R*C)(1000/V)]*D} R = Reaction volume C = Copies/uL V = Volume of virus ...by ddPCR begins with diluting the virus. It is important to note that the dynamic range of the ddPCR is...PCR plate. Maintaining a high droplet count is important for calculating the concentration of your sample...separation (Witte et al., 2016). This is particularly important if your amplicon is longer than 150 base pairs...

...overhangs Cas13a (C2c2) 2x HEPN crRNA RNA 3' A, U, or C (not required by all orthologs) Specific RNA cleavage...spacer sequence and consists of a single A, U, or C base pair. In bacteria, once Cas13a has recognized...of interest and an inactivated fluorescent RNA reporter. If the target sequence is present in the pool...activity of Cas13a becomes activated and the RNA reporter will be cleaved resulting in activation of the...in the field and as always, we appreciate the opportunity to share these invaluable reagents with the scientific...

...fluorescent proteins (GFP and YFP) and protein tags (c-MYC and HA). We have also created the Monoclonal ...repositories, such as Addgene and the DSHB, play an important role in scientific exchange. In addition to ensuring...provide standardized quality-control and technical support to the community. Thanks to Our Guest Blogger! ...

...https://doi.org/10.1096/fj.01-0995fje. Tan, E., Chin, C. S. H., Lim, Z. F. S., & Ng, S. K. (2021). HEK293 ...doi.org/10.3389/fbioe.2021.796991. Urabe, M., Ding, C., & Kotin, R.M. (2002). Insect cells as a factory ...of production, saving about a week of work. An important note is that this is only possible because baculoviruses... will come down to which differences are most important to you — virus type, cost, time, or reagents and...

...construct. Reactions are often cycled between 37°C and 16°C to provide optimal reaction temps for both the... Golden Gate assemblies feature an inverse proportionality between the complexity of the assembly (number...The future of assembly DNA assembly methods are important tools for many areas of science, and researchers...

...our example later, histones) ChiRP-seq RNA Hi-C DNA (genome-to-genome interactions) DNase-seq DNA...commonly think of DNA as a linear polymer of A,T,G, and C nucleotides arranged in a double helix. People are...genome. But not all of those 13,000 regions are important for maintaining ESCs. So which of these 13,000...next-generation sequencing to measure the relative proportion of guide RNAs (gRNAs) across the population. ...

...tag to it. The FLAG, hemaglutinin antigen (HA), and c-myc tags have been the workhorses of the affinity ... tags, a His tag can be fused to either the N- or C-terminus of a protein. Unlike other epitope tags –... Protein tags are usually smallish peptides incorporated into a translated protein. As depicted in the... are available, but scientists would need to incorporate their recognition sites into their protein tag...

...into an attL-containing entry vector.   Method C: Restriction cloning of a restriction enzyme fragment...attL-containing entry vector.   Figure 5: Method C to create an entry clone: Restriction cloning the ...coding sequence. If you choose this strategy, it’s important to include the proper protein expression elements...Clone When making the expression clone, it is important to choose the destination vector that best fits...

...create an N- or C-terminal fusion based on your experimental needs. Equally important, before cloning ... retrieval sequence only functions at the extreme C-terminus [3]. You must therefore determine your cloning...potentially affect function [5]. We and others have reported that FP cysteine residues form inappropriate disulfide...

...gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish Xenopus CRISPR Resources ...which a reverse transcriptase (RT) is fused to the C terminus of Cas9 H840A nickase. The fusion enzyme ...the PBS of the pegRNA and the desired edit is incorporated into the DNA by reverse transcription. The edited...

... a AAV8 expressing eGFP under the human ubiquitin C promoter. Anatomical abbreviations: Gr= Gracile tract...Deisseroth. (2014) Nat Protoc., 9, 1682-1697. Pubmed. C. Soderblom, D.-H. Lee, A. Dawood, M. Carballosa, et...addition, the use of viruses allows much better spatiotemporal control over labeling methods as compared to...

...similar nuclease folds found in other proteins. The C-terminal domain (CTD) of the protein also belongs ...https://doi.org/10.1038/s41586-022-04470-1 Cofsky, J. C., Soczek, K. M., Knott, G. J., Nogales, E., & Doudna...Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., & Doudna, J. A. (2014). DNA interrogation by the...it is limited to only 20 bases, or why it’s so important to choose a target sequence with a nearby protospacer-adjacent... lobes laying open like a book (Figure 2). The portion of the CTD that recognizes the PAM is so flexible..., but they improve cutting efficiency and are important for robust activity in cells (Jiang & Doudna, ... guide RNA stabilizing the DNA-binding protein portion of the CTD, ready to check for PAM sequencines....