...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...types makes them useful models for developmental processes and promising therapeutic tools. The two unique...

...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...resource that provides summary information on Class 2 Cas enzymes. The information is organized in wiki ...this content and much, much more.     Fig. 2 – List of resources and properties for a Cas entry...Database: A Functional Classification System for Class 2 CRISPR-Cas Enzymes [Preprint]. Biochemistry, Biophysics...non-CRISPR-associated host factors required for CRISPR processing. For each enzyme, it will also show you how multiplexed...

... TagBFP 399nm/ 456nm histone 2B (H2B) Chromatin 2 Cerulean 433nm/ 475nm Lyn-tag (derived from the tyrosine-protein...based (chaining-based) cloning. The modules (Figure 2) are contained in individual plasmids and are each...between various cloning methods are listed in Table 2. For example,  chaining-based cloning methods require...suitable for combining endogenous sequences. Table 2: High Throughput Cloning Strategies Method Technology...PMID: 20205762. PubMed Central PMCID: PMC2822740. 2. Engler C, Kandzia R, Marillonnet S. A one pot, one...various mammalian promoters), the same cloning procedure can be used to incorporate each element into the...and protein localization, as well as cellular processes like gene expression and cell migration (Sladitschek...

...question: C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1,...however cutting efficiency of Cas13a is reduced when 2 mismatches are present. The protospacer flanking sequence...PMC5127784. Find plasmids from this paper at Addgene. 2. East-Seletsky, Alexandra, et al. "Two distinct RNase... and functional characterization of diverse class 2 CRISPR-Cas systems." Molecular cell 60.3 (2015): 385...specific RNAs out of a pool of RNAs) or studying RNA processing in live cells. Indeed, groups have utilized dCas13...RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection." Nature 538(7624) (2016):270...

...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...fidelity and bias profiling." bioRxiv (2018): 322297. 2. Potapov, Vladimir, et al. "A single-molecule sequencing...indeed possible, with correct, in-frame assembly proceeding in 99% of 12-fragment assemblies and over 90%...

...study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a minigenome?   The EBOV ...transcription by the EBOV polymerase (reviewed in 2, 5). The minigenome is capable of being replicated...nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola...as it would for a full-length viral genome (Figure 2). While it is technically possible to encode all four...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...systems allow researchers to investigate viral processes in extreme detail. The mechanism of a particular...

...Introduction from Tyler 1:13 - 2:02 : Niroshi and Ben introduce themselves 2:03 - 3:22 : How did you end ...things get tough in the dreaded third year. Find Part 2 Here! We'd like to thank Niroshi and Ben for taking...Future of Research Looks to Improve the Research Process  Browse our Career Posts Resources on Addgene....

... used. Follow the manufacturer’s instructions.   2. Isolate Your Insert and Vector by Gel Purification...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create...containing the ligation mixture with backbone alone; 2. Transformation containing ligation mixture with insert...colonies than the vector + insert plate (unsuccessful 2), or roughly equivalent numbers of colonies on each... or equivalent to backbone + insert, unsuccessful 2 and 3 above), you can try ligating the recipient plasmid....   Overview of the restriction cloning process. Both the plasmid (blue, backbone) and the DNA ... beginning the restriction digest and ligation process, you should carefully choose your backbone and ...

...imaging of diverse tissues. Nature Protocols, 17(2), Article 2. https://doi.org/10.1038/s41596-021-00644-9 ...directly from the Zenodo dataset.     Figure 2: Screenshot showing reagent entries with associated...Knowledge-Base, seeking to create an iterative process that is constantly becoming better, with more data...spatial analysis of cells in complex tissues. Proceedings of the National Academy of Sciences, 117(52),...

...use 2 different secondaries for your antibody pair and visualize both targets in parallel (Fig. 2). For...successfully visualize both proteins.     Figure 2: Two primary antibodies with the same isotype can ...    Figure 1: In the isotype conversion process the variable regions of a parental antibody (blue...isotype (purple, mouse). Depending on the cloning process, all constant regions, just the heavy chain constant...isotype to that of a different group (Fig. 1). This process, called isotope conversion, gives users the flexibility...addition to increasing multiplexing capabilities this process is frequently used to reduce immunogenicity and...therapeutic potential of antibody medicines. In this process, antibodies derived from non-human species like...

...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....

...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... 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...

...screen using CRISPR libraries is a multi-step process (see Fig. 2). In most cases, CRISPR libraries are provided...targeting a specific gene within the genome (see Fig. 2). Individual gRNAs are designed in silico using publicly...mouse, human, fly, E. coli, and T. gondii genes.  2) What genetic modification are you trying to make?... is included on the gRNA-containing plasmid, or a 2-plasmid system in which Cas9 must be delivered separately...genes or entire pathways in a particular disease process. While CRISPR is certainly not the first means ...

...plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR ...then packaged into AAV’s protein shell, or capsid; 2) the pHelper plasmid which expresses adenovirus genes...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human ...therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2. Choi, V.W., Asokan, A., Haberman, R.A., & Samulski...host’s polymerase, but requires Rep proteins to process a double-stranded intermediate into the single-...media are harvested and purified. In total, this process takes 4-7 days, not counting the time needed to...iodixanol and concentrate the purified virus. This process can be completed in one long day, or the virus ...

...plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from Li et al., ...1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed the replicon of pCas into a non-temperature-sensitive... pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella...MUCICAT better, it is faster. Using MUCICAT only needs 2 newly constructed plasmids (pDonor and pQCascade) ...rounds of transformation in 30 days. Figure 2: (A) MUCICAT plasmids for multicopy integration. (...pTargetF system.   Figure 1: The editing process begins with the addition of the sgRNA sequence ...curing, providing users with a complete editing process. The MUCICAT system While the above systems are...

...pathway to allow any cloning procedure to be performed using a simple two-step, 2 hr protocol prior to transformation...Primer design Using specific primer design (Figure 2), IVA cloning can be used to perform any plasmid modification... for each type of modification is shown in Figure 2, and is discussed in more detail below. The main requirement...Biotechniques 10.1 (1991): 62-66. PubMed PMID: 2003926. 2. Bubeck, Peter, Monica Winkler, and Wilfried Bautsch...often, it is also a bottleneck in the experimental process. The majority of current cloning techniques involve...be immediately adopted by any laboratory. All procedures are performed using the same three step protocol... greater homology, so performing more complex procedures can be aided by extending homologous sequences...

.... Diagnostic restriction digests are comprised of 2 separate steps: 1) incubating your DNA with restriction...which cleave the DNA molecules at specific sites and 2) running the reaction on an agarose gel to determine...including a 1.2 kb insert. The plasmid was digested with 2 unique enzymes (HindIII and BamHI) and run on an agarose...to serve as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed...verification is not just necessary but also an easy process. Please vist Addgene's resource for plasmid verification...

...to (1) run a DNA gel to confirm fragment size and (2)  incubate with RNAse and Proteinase K to purify the...proteins of interest to any DNA they were bound to (2) fragmented the DNA (3) used antibodies to specifically...adjustments, to get everything just right!  Fig. 2: Goldilocks is actually celebrating a successful ChIP...the ChIP process and not the upsteam sample collection process or the downstream analysis process. And ChIP...faint of heart!  The Process ChIP can be done for either tissues or cells. The process is very similar for...remain attached to the DNA throughout the sample processing and therefore will require crosslinking, which...done by cutting the tissue sample up and then processing with a homogenizer, but bone or especially fibrous...

...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...