We narrowed to 419 results for: SAM-2

...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...confirmed the insertion of the Lambda insert at the same high frequencies as the antibiotic selection markers...

...genome (Figure 2). While it is technically possible to encode all four viral genes in the same plasmid (as...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...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...

...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... the 3’ end of the target sequence. Figure 2: Effects of (A) double and (B) single base pair gRNA-target...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227... Find plasmids from this publication at Addgene. 2. Zetsche, Bernd, et al. "Multiplex Gene Editing by...cleavage are 42-44 nucleotides in length, about the same size as Cas9’s, but with the direct repeat preceding...

...use 2 different secondaries for your antibody pair and visualize both targets in parallel (Fig. 2). For...both proteins.     Figure 2: Two primary antibodies with the same isotype can not be used for multiplexed... are the same isotype and therefore cannot be indirectly probed simultaneously on the sample. What to ...because the pair belong to the same isotype and will react with the same secondary. If you were to change...that group. If you use two antibodies that are the same isotype then the secondary antibody will bind to...

...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...you’re working with a tissue sample, you’ll need to homogenize your sample before moving on to the next...Homogenization for a tissue sample can also need significant optimization for the sample type and size.) It can...process is very similar for both, though tissue samples do require an extra homogenization step. For this...to focus on the ChIP process and not the upsteam sample collection process or the downstream analysis process...

...backbone alone; 2. Transformation containing ligation mixture with insert and backbone. Sample results indicative... 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...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...between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each...you to either stain your gel after you run your samples or add the stain as the gel is being made (post...

...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...

...detect your targeting vector or gene of interest. 2. Breed your parental mice to generate F1 Once you’...experimental and control mice will have been raised in the same environment and will harbor similar epigenetic modifications...multiple mutations is if the mutations are on the same chromosome. Homologous chromosomes recombine during...two loci, which would get both mutations onto the same chromosome. Until crossing over occurs between the...

...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...not powerful enough for large brain samples. For large brain samples, we recommend pretreatment with ScaleCUBIC1...time points, sacrifice the animals and fix the samples. When you use AAVs, it typically takes a few weeks...excessive amount of XFPs expressed. Then, clear the samples and visualize. For more details on this procedure..."Optical clearing and index matching of tissue samples for High-resolution fluorescence imaging using ...

...two genes over time, at a range of distances from 2-75 Mbp. Labeling multiple, intrachromosomal loci Building...successfully resolved loci with physical map distances of 2 and 75 Mbp, with the calculated fluorescent distances...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the...dCas9 supplied by a separate vector. Figure 2: CRISPRainbow generates 6 unique colors and a white... Find plasmids from this publication at Addgene. 2. Ma, Hanhui, et al. “Multicolor CRISPR labeling of...plasmids from this publication at Addgene. 4. Kiani, Samira, et al. “Cas9 gRNA engineering for genome editing...

...Request review: 1–2 days Prep and QC completed: 6–8 weeks Average shipping time: 1–2 days (USA); 3–4 days... our production process. This process will take 1–2 days, and we anticipate approving the vast majority...produce your Packaged on Request prep and run the same quality control assays we run with all of our in-stock... be 1x1013 and titers for AAV2 to be 7x1012, the same titers found in our in-stock preps. If a request...

..., AAV2/8 has a type 2 ITR and a type 8 serotype. Almost all vectors contain type 2 ITRs, which comes from...differentiated, non-dividing cells.   Figure 2: DNA rescue and transduction of a conventional single-stranded...quantitative PCR-based method for the titration of AAV-2 vector stocks."Molecular Therapy 6.2 (2002): 272. ...episomal and does not divide and replicate in the same way the host DNA does. Because of this, fast dividing...McCarty, D. M., Paul E. Monahan, and Richard J. Samulski. "Self-complementary recombinant adeno-associated...

...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 ...between the sites. If a DNA sequence is floxed in the same orientation, the SSR will excise the DNA fragment...reporter with one loxP site and one lox511 site in the same orientation, and placing the additional loxP and...

...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Wang,...(see maps section for images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre...Digital PCR Method. Human Gene Therapy Methods, 29(2), 96–103. https://doi.org/10.1089/hgtb.2017.198   ...of the viral capsid proteins VP1, VP2, and VP3. Samples of viral preparations are subjected to polyacrylamide...proteins as a fraction of total protein present in the sample is also determined and used to determine purity...shown relative to the total protein present in the sample.   Sterility Viral vector preparations are ...rigorous barcode matching system that follows the sample from the DNA tube all the way to the cleared viral...

...those in Figures 2 and 3 don’t show these regions, even if they are present in the sample. Other techniques...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...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,...

...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...non-targeting guides, when both are analyzed using the same false-discovery rate cutoff.  Adding safe-targeting...genome-wide CRISPR screens are prone to some of the same flaws as RNAi screens, such as non-specific toxicity...

...still accrue on average 2 to 3 times as many citations as other papers in the same journals! So, is all ... 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010 167  Nature 3.3%  10.5%  3.2  534.1 3...Microbiology  1.6% 1.2%  0.75   125.3 In figure 2, you can see the full impact from using these weighted...This post was contributed by guest bloggers Samantha Zyontz and Neil Thompson from the MIT Sloan School... Under such circumstances, we would observe this same correlation, but it wouldn’t be causal. To control...Addgene deposits as if they were published in the same journals as those that did make deposits to Addgene...along the way. Many thanks to our guest bloggers, Samantha Zyontz and Neil Thompson. Special thanks to Addgenies...

... PubMed PMID: 22428977. 2. Choi, V.W., Asokan, A., Haberman, R.A., & Samulski, R.J. (2007). Production...then packaged into AAV’s protein shell, or capsid; 2) the pHelper plasmid which expresses adenovirus genes...takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR run and data analysis. Refer to...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human ...particles that are present in an AAV prep. AAV samples are first DNaseI digested to remove any residual...technology or a TaqMan primer/probe set can be used and samples are quantified by comparing to an AAV standard ...AAV, remember to account for the dilution of the sample for the DNAseI digest. Use good sterile technique...

...Carballosa, et al, (2015) eNeuro, March/April 2015, 2(2) e0001-15.2015. Pubmed. Resources: http://openspim.org...light sheet, typically 2-6  microns, illuminates only one thin plane of the sample surrounding the focal...orthogonal-plane fluorescence optical sectioning (OPFOS) (2). Arne Voie, David Burns and Francis Spelman focused...beam into a thin sheet to illuminate a fluorescent sample and captured the reflected light using a different...laser and the reflected light travel through the same objective lens. Using this method, these authors...problem is to illuminate and acquire images of a sample from two opposing sides and merge the images, such...

...procedure to be performed using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As...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...laboratory. All procedures are performed using the same three step protocol:  Fragment generation - Any...events, all linear fragments need to get into the same cell. For simple cloning requiring just one or two...vector and insert with separate primer pairs in the same PCR. Add homologous sequences to one of the primer...