...44632, the contaminant we mixed into the original AAV sample at 5%. Detecting 5% contamination is a good... Our recombination software looks for and quantifies the presence of recombinants in recombinase-dependent...sequences. Recombinase-dependent sequences may be subject to recombinase-independent recombination during ...containing the suspected contaminating sequence. In most cases, the source of the contamination can be identified...prep we suspect is contaminated. For AAV-68544, the best match for the contaminant is Addgene plasmid ...This recombination produces a minor pre-recombined population that does not require the recombinase for...viral vectors and estimate that recombinase-independent recombination occurs in 0.1-0.6% of AAV particles...

...human kinases remains challenging. Small molecule kinase inhibitors usually target soluble kinase domains...thereby rapidly create desired allele combinations and combinatorial genomic libraries. MAGE has allowed...pORTMAGE) expresses the λ Red recombinase enzymes, as well as a dominant-negative mutator allele of the... HA tagged BioID2 for N-terminal fusions and Myc tagged BioID2 for C-terminal fusions. Kim, et al. Mol... pCAG-T7-TALEN(Sangamo)-Destination series, pCAG-Golden-Gate-Esp3I-Destination Takashi Yamamoto pcDNA-TAL-NC2...Human kinase domain constructs for automated bacterial expression The misregulation of human kinases has...recently compiled the Human Kinase Domain Constructs Kit, a library of human kinase domain constructs developed...

... manipulation. Cre recombinase, originally named because it “causes recombination” (although later referred...cyclization recombinase”), is a 38 kDa protein responsible for intra- and inter-molecular recombination at the...alluded to above, the Cre recombinase catalyzes the site specific recombination event between two loxP sites...separate recombination events? One potential limitation of using loxP sites is the inability to tightly...specific recombination event in a single system. The Saccharomyces cerevisiae FLP-FRT recombination system... 1. Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. Sternberg, N. and.... 2. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Sauer...

...deposited by the Gradinaru lab. For simplicity, in each section we’ll list the Gradinaru capsids first, ...be used to target endothelial cells in the retina and spinal cord (Krolak et al., 2022). Systemic capsids... X., Luo, Y., Einarsson, P. H., Greenbaum, A., Jang, M. J., Deverman, B. E., & Gradinaru, V. (2020). Multiplexed...promoters and enhancers, miRNA target sites, and recombination target sequences, denoted by triangles] facilitate.... In cases where it’s critical to minimize or eliminate off-target expression, it may be better to choose...capsids in our repository are deposited by the Gradinaru lab. You can visit our Caltech Systemic Capsids...

...protein is composed of an n-terminal DNA recognition domain and a mostly c-terminal nuclease domain. Feng Zhang's...Splitting wild-type Cas9 into n-terminal (Cas9(N)-2xNLS) and c-terminal (Cas9(C)-2xNLS) fragments facilitates...with different N- and C-terminal tags. Additionally, by using an adapted destination vector with a heterologous...bearing the "Bunch of Baby Spinach"aptamer, which is a brighter version of Spinach2, an RNA aptamer that fluoresces.... Bunch of Baby Spinach, or BoBS, consists of three tandem copies of the Baby Spinach core embedded in...with Broccoli Vegetable tags, including Spinach, Spinach2, and Broccoli, can benefit your RNA experiments... control of gene knockout or activation, the c-terminal cas9 fragment was fused with FK 506 binding protein...

... Other variants of Spinach include Baby Spinach, a miniaturized version of Spinach that retains similar... Hammond’s group created Spinach2 using site-directed mutagenesis of Spinach. In doing so, they identified...was consequently brighter than Spinach. The initial studies using Spinach2 showed its ability to track RNA...imaging. Bunch of baby spinach, or BoBS, contains tandem repeats of Baby Spinach. It was developed in a...twice as bright as Spinach2 when bound to DFHBI. It’s also smaller than any form of Spinach - only 49 nucleotides...Addgene’s collection. Green fluorescent aptamers Spinach and Broccoli and their derivatives are aptamers..., but not quantitative, studies. Additionally, Spinach, the first aptamer identified to bind DFHBI, is...

...bacteria. In vivo, these recombination reactions are facilitated by the recombination of attachment sites ... in the donor vectors and the destination vectors prior to recombination, and it is exchanged with the...or by Restriction Cloning Destination vector attR sites for recombination; ccdB gene for negative selection...vector systems. With the appropriate entry and destination vectors, one can use Gateway to clone a gene ...vitro version of the integration and excision recombination reactions that take place when lambda phage ...attP) and the bacteria (attB). As a result of recombination between the attP and attB sites, the phage integrates...into the bacterial genome flanked by two new recombination sites (attL-left- and attR-right-, Figure 1)...

... primer on how Cre-lox recombination works. Advances in Cre-lox recombination have included inducible...coupling of Cre-lox with FLP-FRT recombination to generate additional recombination events, and the synthesis...excision results in one of three recombinations, and subsequent recombinations cannot occur. While the Brainbow...Brainbow-1.0 mice.) The combinatorial effect of multiple, independent recombination events leads to a rainbow...systems remain valuable to biology. Cre-lox recombination, discovered in the 1980s, is one of the most... loxP sites to specify distinct patterns of recombination. In 2007, Joshua Sanes and Jeff Lichtman, both...M-CFP (membrane tethered cyan). Without Cre recombination, RFP is expressed. Cre can mediate one of two...

...factor and promoter pairs referred to as binary systems. Binary systems are genetic tools that consist ...repressible element to this binary system (making this a repressible binary system). The GAL80 repressor...expression and largely defines the amount of recombinant protein that will be produced. Many commonly-...default ON to an OFF state, as well as repressible binary systems. In recent years, a number of more complex... previous post. In the past 30 years since the original Tet-Off system was described by Gossen and Bujard...inducible manner detailed in our previous post. While originally developed for use in mammalian cells, this system...consumption phase of yeast culture. Repressible Binary Systems GAL4/UAS In Drosophila or development studies...

...how the Cre-loxP recombination system can be used to induce site-specific recombination events, and that...sites to induce site specific recombination events: Cre recombinase binds loxP sites, while FLP binds ...presence of Cre recombinase.   Once the SSR of choice is introduced, recombination can proceed either...any additional recombination events are impossible even in the presence of Cre recombinase. This plasmid...BL. 2012. Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression...orientation of the flanking loxP sites directs the Cre recombinase to invert, translocate, or excise a DNA fragment...such strategy--the FLEx switch--which utilizes recombination elements to turn off expression of one gene,...

...cytosine base editor by coupling a cytidine deaminase with the inactive dCas9 (Komor et al., 2016). These fusions...RNA adenine deaminases have been engineered and/or evolved to catalyze cytidine deamination instead of ...adenine deaminases. The team used directed evolution to create one from the RNA adenine deaminase TadA.   ...protein (catalytically dead or nickase) fused to a deaminase that makes the edit A gRNA targeting Cas to a ... and team wanted to force the cell to use the deaminated DNA strand as a template. To do so, they used...copying the base edit. Figure 2: Cytidine deamination takes place on the free strand of DNA and converts...created the Target-AID base editor using a cytidine deaminase from sea lamprey fused to Cas9 nickase (Nishida...

...protein genes, Cre recombination is required before fluorescence can occur. Cre recombinase can be used alongside...only expressed following a recombination event. Tissue-specific Cre recombination in this mouse generates...may acquire one of a variety of colors when a combination of XFP are expressed. Each cell remains the same...with Brainbow The Lichtman group developed the original iteration of this technology in 2007. Used to ...cells will fluoresce either red or orange until recombination occurs. Following the development of Brainbow...the default state of the transgene such that recombination is necessary to excise the non-fluorescent YFP...that express the construct but do not undergo recombination can be detected by immunostaining of the truncated...

... Keep your contaminants away While it’s likely that you’re the main source of contamination for your experiments... their original condition and can replace your working stock if it gets too old or contaminated. This ...hopefully help you keep a well organized lab and contamination free environment for successful experiments:... up to half an hour can help keep your hood contamination free, but you should keep in mind that UV light...precautions necessary to keep your cells from becoming contaminated. Wipe down everything that’ll be brought under...entering the hood is another great way to prevent contamination. This sounds rather trivial, but often solves...is in progress.Learn more about cell culture contamination in this post from the ATCC Declutter! Keep the...

...for authentication and contamination) and cell lines can become cross contaminated and are invariably much...still used today to test for HeLa contamination. HeLa contamination is still a problem today. It is now...cell lines suffer from contamination with HeLa being the most frequent contaminant, closely followed by ...different researchers are free of contamination and match their original description. The oldest, largest...Unreproducible results are rife. Misidentified or cross-contaminated cells have been known about since 1966 when ...the majority in those days) were in fact all contaminated with HeLa cells, the first immortalized human...wasted in basic oncological research due to contamination are in the tens or hundreds of millions, let...

...machinery making an original C-G pair converted to T-A or reverted to the original C-G by base excision...invented CRISPR-base editors by tethering cytosine deaminase with Cas9 nickase (nCas9). nCas9 creates a nick...indel formation. After binding with DNA, the CBE deaminates a target cytosine (C) into a uracil (U) base...., there are no naturally known 'DNA adenosine deaminases' which would theoretically give rise to an adenine...resulted in the successful evolution of an adenosine deaminase that can act on DNA for adenine base editing. ...created a mutant library of E. coli tRNA adenosine deaminases (ecTadA), fused it with dCas9, and transformed....          The artificially evolved adenosine deaminase catalyzes the conversion of a target ‘A’ to ‘I...

...site-specific serine recombinase. First, the prime editor installs the recombinase “landing sites,” then... MagIC-Cryo-EM for enrichment and structure determination LEA proteins protect samples during cryo-EM ...editing CHARM: A compact epigenetic silencer Recombinant antibodies for marking astrocytes MagIC-Cryo-EM...MagIC-Cryo-EM for sample enrichment and structure determination By Mike Lacy Conventional cryogenic electron ...MagIC-cryo-EM for sample enrichment and structure determination. A) Schematic of workflow. B) Detail of spacer...Funabiki, H. (2024) MagIC-Cryo-EM: Structural determination on magnetic beads for scarce macromolecules ...then the recombinase handles the installation of the donor sequence. The latest work evolves and engineers...

...phosphoserine into recombinant proteins, such as the kinase MEK1 (5). MEK1 is inactive without requisite...candidate regulatory kinase is known, it’s possible to generate non-phosphorylated recombinant protein and perform...in vitro kinase reactions. Often, however, researchers lack knowledge of the regulatory kinase, access ...access to active kinase for the reaction, or have difficulties with kinase specificity in vitro. The establishment...establishment of a system in which purified, recombinant protein could be produced with site-specific incorporation...incorporation of phosphoserine directly into recombinant proteins (5). Optimization of this system defined...create a pSer/phosphomimetic dual mutant) elevated kinase activity over 70-fold over dual phosphomimetic ...

...first segments of the spinal cord showing the cortico spinal tract, CST, spinal cord and cranial nerve...Cuneate tract, dCST= dorsal cortico spinal tract, vCST= ventral cortico spinal tract, dc= decussation of the...was first pioneered by Voie and colleagues and originally named orthogonal-plane fluorescence optical sectioning...Spelman focused a laser beam into a thin sheet to illuminate a fluorescent sample and captured the reflected... lens oriented perpendicular to the plane of illumination (i.e. light sheet).  This is in contrast to ...described the single-plane or selective-plane illumination microscope (SPIM). In order to improve axial...optical sectioning of a specimen is based on discriminating the out-of-focus reflected light by using a...

...will not be gel purifying it, you may need to inactivate the enzyme(s) following the digestion reaction...

...Blog Posts Antibody Guide Western Blot Protocol Recombinant Antibody Purification Protocol Introduction Immunocytochemistry...