...kinetics. For example, the light-gated cation channel, Channelrhodopsin 2 (ChR2), exists in several states:...different steps and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail,...stimulation         In the video above, Channelrhodopsin 2 (Chr2) has been expressed in the motor cortex of a...lasers are relatively cheap and will run you about $2-3,000/laser. However, an important caveat is that ...PMID: 21628464; PubMed Central PMCID: PMC3138296. 2. Sparta DR, et al. Construction of implantable optical...board, Jacqueline N Crawley  [et al]. 2012;Chapter 2:Unit2 16. doi: 10.1002/0471142301.ns0216s58. PubMed...There are several important considerations with fabrication of the fiber optics needed for optogenetics. ...

...Table 2 for more details on the effect of pH on the fluorescent properties of Rosella.   Table 2: Effect...in yeast. Autophagy,4(2), 205-213. doi:10.4161/auto.5331. PubMed PMID: 18094608 2. Sargsyan, A., Cai, J...environment like the vacuole or lysosome. See Table 2 for a summary of what color fluorescence Rosella emits...fluorescence accumulates in the vacuole. See Figure 2 for an example of what fluorescence looks like for...radioactivity in the culture supernatant (this is indicative of protein degradation). To control for protein...studying autophagy. This method relies on the identification of autophagic structures based on morphology...

..., Huebener , Rose 83899 pAAV-mDlx-GCaMP6f-Fishell-2 Dlx GCaMP6f none Constitutive 1, 9, rg* Fishell 100833...pAAV.CAG.GCaMP6f.WPRE.SV40 CAG GCaMP6f none Constitutive 1, 2, 9 Kim , GENIE 100837 pAAV.Syn.GCaMP6f.WPRE.SV40 Syn...pAAV-CAG-NIR-GECO2G CAG NIR-GECO2G none Constitutive 1, 2, 9 Campbell Calcium Sensor: HaloCaMP1a 138327 pAAV-synapsin-HaloCaMP1a-EGFP...offers you even more options. Select from a vast catalog of eligible AAV plasmids and we’ll make the viral...

...left free to interact with the target DNA. Figure 2: Overview of the NHEJ repair mechanism Cas9 will only...systems enable researchers to target anywhere from 2–7 genetic loci by cloning multiple gRNAs into a single...Cas9 is included in the gRNA-containing plasmid, or 2-vector systems, in which Cas9 must be delivered separately...your experimental cell population (Figure 8E). In a 2-vector system, you’ll need to either co-infect with...the presence of infectious organisms (like SARS-CoV-2 ) and genetic mutations. Similar to Cas9 and Cas12...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...off-target effects by using a single Cas9 nickase and 2 different gRNAs, which bind in close proximity on ...

...them in Regenerative Medicine. Acta Naturae, 2010. 2(2): p. 18-28. PubMed PMID: 22649638. PubMed Central...capability to differentiate into specialized cell types [2]. We general speak of 4 levels of stem cell potency...zygote through to the morula stage are totipotent [2-4]. Pluripotency: When a cell has the ability to differentiate...stem cells (ESCs), which I discuss in detail below [2]. Multipotency: When a cell can differentiate into...PMID: 26726264. PubMed Central PMCID: PMC4686367. 2. Jaenisch, R. and R. Young, Stem cells, the molecular...stem cells are: Their ability to self-renew or replicate while maintaining the undifferentiated “stem” ...Unipotency: As the name suggests, cells under this category can differentiate into only one cell type. Muscle...

...Each antibody catalog item is tested for shipping stability by incubating at 37 °C for 2 weeks, freezing...recommended applications. The specific QC experiments performed varies for each antibody catalog item. Whenever...specific antibody can be found on the antibody's catalog page or by contacting [email protected] . Some of...Innovation (IPI). For these antibodies, IPI will be indicated as the Depositing Lab on the antibody webpage....cells and will interfere with conjugation. For applications such as these, the buffer can be removed by ...

...Schmid-Burgk, J.L., et al (2). In this post, I’ll cover two great applications of the NLuc technology. The...GpNLuc and OgNLuc are available through Addgene (Cat.# 70185 and 70186, respectively). Antares Chu, J...construct for Antares is available through Addgene (Cat. # 74279). Enhanced Nano-Lanterns  Suzuki, K., et...molecularly evolved from mNeptune2 through 33 mutations and 2 deletions. CyOFP1 has a higher quantum yield, brightness...luciferin 24 hours later demonstrated a more than 2-fold brighter signal from Antares as compared to Fluc...PMID: 22894855. PubMed Central PMCID: PMC3501149. 2. Schmid, J.L., et al. (2016) CRISPaint allows modular...fluorescence. The paper reports a wide variety of applications of the Enhanced Nano-Lanterns including monitoring...

...researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a minigenome...minigenome replication and reporter gene transcription by the EBOV polymerase (reviewed in 2, 5). The minigenome...template for replication and transcription, as it would for a full-length viral genome (Figure 2). While it...nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola...500 ng NP, 500 ng VP35, 100 ng VP30, 100 ng L, and 2 μg minigenome plasmid per well (in a 6 well plate ...PubMed PMID: 9847328. PubMed Central PMCID: PMC103829. 2. Conzelmann, K. K. "Reverse genetics of mononegavirales...Technical Tips). Minigenome replication and transcription In order to replicate the minigenome, the viral...

...Figure 2: Effects of (A) double and (B) single base pair gRNA-target mismatches on modification by AsCpf1...class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227. Find plasmids from this publication at ...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Figure from Zetsche et al., 2015...2015. How was Cpf1 found and tested? Class 2 CRISPR systems, including the type II Cas9-based system...employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR from Prevotella and Francisella...at Addgene. 2. Zetsche, Bernd, et al. "Multiplex Gene Editing by CRISPR–Cpf1 Using a Single crRNA Array... this publication at Addgene. Makarova, Kira S., et al. "An updated evolutionary classification of CRISPR-Cas...

...Originally published Dec 7, 2017 and updated Jul 2, 2020. Promoters may be the star of gene regulation...sequence targeted in the experiment. Figure 2: Comparing CAPTURE and CAPTURE 2.0.     Original...cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or more gRNAs Two separate lentiviruses...delivering 1) a BioTAP-tagged dCas9 and an eGFP tag, and 2) gRNAs Number of loci targeted One Many Sample...identified and the level of on-target enrichment. 2. Determining the spatial and hierarchical organization...Cytogenetics, 11. https://doi.org/10.1186/s13039-018-0368-2  Liu X, Zhang Y, Chen Y, Li M, Zhou F, Li K, Cao H... gene’s transcription. Sometimes enhancers are located thousands of base pairs away from the gene they...

...for the 2+ million DHS sites. See below for further details. DNA is a 3D Fractal Figure 2: DNA compaction...: 1. Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the ...NANOG enhancers 3. CRISPR out the enhancers from step 2. See which ESCs die or differentiate Step 3 reveals...expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt these enhancers...to CRISPR out the enhancers we identified in step 2. The result is a heterogenous population of stem cells...helps us to locate interesting addresses in the genome. Let’s now examine how we can locate powerful stretches..., start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning of the ...

...University of Manitoba due to their Biosafety Level-2 or 2+. Please Note: Despite the fact that AAVs themselves...important factors related to location. Location (1): What tissue? Location (2): Where in the tissue/which...long and stable expression (up to 9 months in mice [2]). Low Immune Response: Compared to other viral vectors...may be sufficient to overcome the transport issues. 2. Failure to plan (for storage) Is planning to fail...Find Ready-to-Use Retrograde AAV Preps at Addgene 2) Comparison of genetically engineered receptors (i.e...Therapy 15.10 (2007): 1740-1743. PubMed PMID: 17882145. 2. Zincarelli, Carmela, et al. "Analysis of AAV serotypes... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain." PloS one 8.9 (...

... can be used to diagnose the presence of SARS-CoV-2 RNA, but inadequate access to reagents and equipment...named this system SHERLOCKv2 for SHERLOCK version 2. Here are some of the improvements: SHERLOCKv2 uses... a protocol for using SHERLOCK to detect SARS-CoV-2 RNA. The test is started using RNA purified from patient...recently adapted the DETECTR platform to detect SARS-CoV-2 using a lateral flow strip format. They published ...CY (2020) CRISPR–Cas12-based detection of SARS-CoV-2. Nature Biotechnology. https://doi.org/10.1038/s41587... and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Molecular Cell 60:385–397 . https...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi....

...transient mammalian expression 101126 pACP-tag (m)- 2 Vector ACP/MCP-tag Empty backbone for stable and transient...compatible with multiple applications (different fluorophores, biotin, bead purification, blocking agents) ACP...formation in nonribosomal peptide biosynthesis. Catalytic role of the condensation domain. J Biol Chem. ...

...that have been developed into retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses...effectively. Retroviral structure   Figure 2: Structure of a retrovirus. Image re-used from Dong...Dong & Kantor, 2021 under CC-BY license. Figure 2 gives a detailed view of the structure of a retroviral...transfer of RT, which would actually occur in step 2 during uncoating. After uncoating, reverse transcription...one of the two strands’ DNA counterparts from step 2 will be integrated by the viral integrase protein ... Air. Environmental Science & Technology Letters, 2(4), 84. https://doi.org/10.1021/ACS.ESTLETT.5B00050...retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, we will mostly use the gamma-retroviruses...

...and Amy tell us a little about themselves. Chapter 2: What is Science in the News (SITN)? - SITN is a graduate... to be crazy complicated! Listen to the full podcast for more great science communication tips or listen...tons of ways you can get involved in science communication. In this second half of our conversation with...many things you can do start your own science communication student group and get more involved with your... graduate student group with the goal of communicating science to the general public and training the ...the next generation of science communicators. In this chapter we learn about some of their events - science... peer-to-peer feedback in effective science communication. Chapter 7: The Goal of the SITN Podcast - Vini...

...fluorescent state make them useful for 2-color imaging with green PA-FPs. Table 2: Properties of Selected Reversible...conventional FP imaging. PA-FPs are divided into 2 main categories - those with irreversible and reversible photoactivation... forms of the chromophore which contribute to the 2 peaks of wtGFP’s excitation spectrum- a major 397 ... / N99K / S148L / I165V / Q167P / L169V / I203R) (2) and PA-mRFP1(S146H / I161V / I197H) (3) were derived...with two distinct wavelengths of light (see Table 2). The best known reversible PA-FP is the naturally... above background. The majority of PA-FPs come in 2 flavours- monomeric and tetrameric. Tetrameric PA-...most PA-FPs, but, in 2015, the Looger Lab designed 2 variants of EosFP that can withstand these conditions...

...Golden Gate Assembly is commonly used to assemble 2–10 DNA fragments in a single “one-pot” reaction to...the transformants onto the selection plate. Figure 2 shows representative transformation plates obtained...fidelity studies conducted by Potapov et al. at NEB (1, 2). While T4 DNA Ligase, the mainstay of most biotechnological... (percent correct) Calculated colony totals Per 2 μl assembly reaction Per full assembly reaction** ...calculated yields from entire outgrowth built from 2 µl of the assembly reaction, and from the entire assembly... bias profiling." bioRxiv (2018): 322297. 2. Potapov, Vladimir, et al. "A single-molecule sequencing assay...upon growth on LB/Cam/X-gal/IPTG agar plates, indicating successful reconstruction of the coding sequence...

...resulting from non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation...each sample can be quickly calculated (details in [2]) and can be used to quantify rates of mutation in... have been reported for mismatch nuclease assays (2, 6, 7). Ability to distinguish between mono-allelic...assays, in addition to testing their performance (2). Remember that although digital PCR is a great screening...PMID: 24509632. PubMed Central PMCID: PMC4063274. 2. Findlay, S. D., Vincent, K. M., Berman, J. R. & Postovit...a gene of interest is one of the most common applications of precision genome editing, the remainder of...single molecule resolution of digital PCR allows classification of individual target molecules based on their...

... H. J. (2009). Rats! Disease Models & Mechanisms, 2(5–6), 206–210. https://doi.org/10.1242/dmm.002733 ...studying genetic modification for their ease of manipulation and their ability to be replicated. Because E...., 2004). Additionally, breakthroughs in gene modification tools and methods such as CRISPR have vastly...Escherichia coli as a Model Organism and Its Application in Biotechnology. In Escherichia coli - Recent...Physiology, Pathogenesis and Biotechnological Applications. InTech. https://doi.org/10.5772/67306 Koornneef... bacteria with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-04899...