We narrowed to 513 results for: gats

...SpCas9. xCas9 has a PAM requirement of NG, GAA, or GAT, making it a more flexible cutter with the added ...

...everything outside the gate is a different population.  Gating and plot orders  As the gates progress from broadest...order you use them in, is known as your gating strategy.  Gating Strategies   The series of markers you...This shape is called a ‘gate’; it tells the machine that everything inside the gate is one population and...identified and isolated.  Following a gating strategy Figure 4 shows a gating strategy from a published experiment...differentiate cells from debris. Cells are gated, and in the gated population, two types of FSC are used to...decided to place their gates on the plots! Flow data is messy, so learning how to gate takes a lot longer ...numbers by gated populations; this indicates the percentage of total events contained in the gated population...

...simply “dump” them by gating on the negative population.     Figure 2: Dump gating labels all unwanted...usually conjugated to a fluorophore, not every marker will have a corresponding fluorophore-conjugated antibody...cells of interest via FACS using negative selection is called dump gating. Let’s say we want to sort out...cells. As a result, the largely negative live cells can still be easily gated on during the analysis. Conclusion...surface, dump gating is a useful way to get rid of undesired cell populations. Dump gating uses the same...for different markers, making it easy to gate on the negative cell population. To detect non-surface markers...Harvard Medical School. His projects include investigating negative regulators of T cells and cytokine signaling...

...directly conjugated detection antibodies or unconjugated detection antibodies followed by conjugated secondary... antibody can be directly conjugated with a reporter enzyme or a conjugated secondary antibody can be ...available and appropriately conjugated antibody. While labs can conjugate antibodies themselves, the process...components can interfere with conjugates. Make sure to do your research before conjugating at the bench.   Indirect...indirect method uses both an unconjugated primary antibody and a conjugated secondary antibody. Incorporating... primary antibody does not need to be conjugated, and conjugated secondary antibodies are easy to find...antibody may either be conjugated directly with the reporter enzyme or used with a conjugated secondary antibody...

...highly active in nick ligation and DNA end-joining, but will also efficiently ligate many undesirable structures...T4 DNA Ligase, can ligate nicks containing one or more mismatches near the ligation junction with high...side of the ligation junction (the base pair providing the 3 ́-OH terminus to the ligation) than on the...the base at the ligation junction. Thus, all four bases at either side of the ligation junction were represented...plasmid construction (restriction cloning), adaptor ligation for next generation sequencing (NGS) library construction...commonly utilized in vitro, Taq DNA ligase will ligate only nicks (5-8). Taq ligase is a NAD+-dependent... for others, there is a need for the exclusive ligation of fully base-paired nicks with no gaps. For example...

... useful for the ligation step. 3. Ligate Your Insert into Your Vector In the ligation step, you mix your...to insert. It is critical to set up a negative control ligation reaction with no insert DNA added. This...transformations after a ligation: 1. Control Transformation containing the ligation mixture with backbone...successful and unsuccessful ligations are indicated below. A successful ligation will have few colonies on... in the ligation reaction. 5. Purify the finished plasmid   Once it looks like your ligation has worked...When cloning by restriction digest and ligation, you use restriction enzymes to cut open a plasmid (backbone... Before beginning the restriction digest and ligation process, you should carefully choose your backbone...

...to carry out a pooled set of ligations in an hour or less. The ligations combine the fragments in an order...restriction digests, gel purifications, or ligations. The general Gateway cloning protocol is fairly flexible...prevent any non-recombinant vectors from propagating. The ligation-independent methods like Gibson and SLIC...Golden Gate assembly allows one to simultaneously complete an ordered set of scarless ligations following... demanding results. If you’re in a hurry, Golden Gate cloning might be for you. This methodology relies... These fragments can be pooled in one tube and ligated in as little as 30 minutes! Alternatively, you ...overhangs in the backbone. Once combined, the cut and ligation processes happen in a matter of minutes. Cost...

...the Ly6C negative population from the Ly6C positive population. Figure 2: An FMO gating example....in the later gates, are more likely to require FMOs. Isotypes Isotype controls are negative controls that... They are made up of compensation beads and a conjugated antibody. Compensation beads are a mixture between...between synthetic beads that can bind to a conjugated antibody and beads that are unable to bind to them...beads create an artificial positive (bound) and negative (unbound) population. The benefit of beads is ...room on the plot to see all of your positive and negative cells. This can be achieved by adjusting your ...stands for fluorescence minus one. It is used as a negative control for a specific marker in your experiment...

...system combining the advantages of Golden Gate and Multisite Gateway cloning methods developed by the lab ...constructs. As the name suggests, GreenGate is based on the Golden Gate cloning method, but has been modified...component plasmids from building blocks via Golden GATEway Cloning Frustrated by the complexity of assembling...just the set of tools that you need. The Golden GATEway cloning kit simplifies the cloning process for ...sequences for a desired component are cloned into Golden Gate entry vectors via traditional restriction enzyme...oligonucleotides. These entry vectors are used in a Golden Gate cloning step to assemble the individual components...the destination vectors are used with Multisite Gateway cloning to generate the final construct. Up ...

... to propagate because CcdA wasn’t available to counteract the toxin. If, however, an investigator were...recombinant plasmid to propagate. Any cells that contained non-recombinant vectors (re-ligated empty vectors, ...ccdB aspect, Gateway takes advantage of the same principle that cells will not propagate while expressing...recently developed Gibson Assembly Cloning and Gateway® cloning technologies, as well as several others...focus on a unique gene that makes the popular GatewayTM method possible: ccdB. But what is ccdB, what ... to thoroughly screen colonies for the insert. Gateway® technology (developed by InvitrogenTM) is essentially...insertion will completely replace ccdB with the investigator’s insert of interest. Hence correct clones are...

...cells are passed through the path of a laser and interrogated with various visible and fluorescent light sources...path of the laser in single-file where it is interrogated by the optical system (Figure 1).   ...a variety of fluorescent light sources. When interrogated with a fluorescent light source, a cell expressing...expression, the further the shift of a cell from the negative control, the higher the level of expression. A...  Protein expression, measured using antibody conjugated fluorophores Fluorescent antibodies get around...staining, the primary antibody against a target is conjugated to a fluorochrome. In indirect staining a primary...antibody binds to a target and a fluorochrome-conjugated secondary antibody binds to the primary antibody...

... present in the gate.   Figure 1: Example of yes-no flow plot of the (negative) control (left)...MFI, you can still continue gating populations of interest with normal gates, just based on a histogram...way, the GFP- population should overlap with the negative control population (Figure 1). Based on where ...where the GFP- population ends, you can draw a gate which selects anything to the right of it, marking it ...left) or with the sample (right). Negative and positive samples are clearly distinguishable as two separate...treatment processes, you may see a shift in the negative population, so it no longer overlaps with the ...from Figure 2 that determining where to place your gate based on the dot plot would be complicated.  ...

...methods such as Golden Gate or Gibson assembly. Golden Gate Assembly of gRNAs Golden Gate Assembly uses Type...needed for the final ligation step. Plasmids are then digested using BsmBI and ligated into Cas9 or dCas9...lab. Scaling up your multiplexed gRNAs with Golden Gate and Gibson assembly methods If you want to scale...destination vector. The first step in CRISPR/Cas9 Golden Gate multiplexing is to clone the oligonucleotides specifying...the procedure will vary slightly.   Two Golden Gate options that are available from Addgene follow these...repression), and Fok1-dCas9 (dimeric nuclease). Gateway assembly method Frew Lab Multiple Lentiviral Expression...should be compatible with type IIS enzyme BfuAI. Gateway cloning is then used to combine the multiple gRNAs...

...RepB) necessary for replication in various hosts (Pagatto and Dillon 2001).  The plasmid has its own replication...self-transmissible, meaning that the plasmid is conjugative and carries the genes necessary for transfer ...with Shown to work in IncN pCU1 Gram-negative  Agrobacterium spp., Bradyrhizobium spp., Rhizobium...Rhizobium spp. IncP RK2 Gram-negative  Achromobacter parvulus, Acinetobacter spp., Aeromonas...campestris IncQ RSF1010, R300B, R1162 Gram-negative and Gram-positive  Acinetobacter calcoaceticus...campestris, X. maltophilia IncW pSa, pR388 Gram-negative  Acinetobacter calcoaceticus, Aeromonas liquefaciens...Zymomonas mobilis pBBR1 based pBBR1  Gram-negative  Alcaligenes eutrophus, Bartonella bacilliformis...

...in combination of two conjugated single fluorophores, like APC-Cy7. Conjugating Cy7 to APC results in ...antibody is conjugated to. Since CD45 is a common marker, finding an antibody already conjugated to my fluorophore...Harvard Medical School. His projects include investigating negative regulators of T cells and cytokine signaling...proteins is through treating cells with fluorophore-conjugated antibodies that attach to a specific target. ...whether that fluorophore appears alone or in conjugation with another fluorophore. The brighter a fluorophore...fluorophore when excited by the red laser. When conjugated to Cy7, however, (APC-Cy7) the brightness changes...fluorescent reporters, such as GFP, and fluorophore-conjugated antibodies together in the same color panel. ...

...buffer components are compatible with your conjugate and conjugation chemistry.  Quantification Once you’ve...antibodies to their parental hybridomas. Aggregation and storage Aggregation of antibodies may impact their quality... used for recombinant antibody production is Komagataella phaffii (a.k.a  Pichia pastoris) because it ...concentration using these columns. If you are conjugating your antibody in a downstream step, you’ll want...quality and efficacy (Wang et. al., 2018). Aggregation can arise from a shift in temperature, pH, or salt...concentration (Amin et. al., 2014). Antibodies may even aggregate just from dropping the tube they are contained... PMID: 25819219. Wang W, Roberts CJ. Protein aggregation - Mechanisms, detection, and control. Int J Pharm...

...not the other. 3) The beads are collected by centrifugation or a magnet. 4) Unbound proteins are washed...Protein G, or the recombinant Protein A/G are conjugated to agarose or magnetic beads and allowed to bind...target protein. The beads can be collected by centrifugation (agarose beads) or a magnet (magnetic beads... cases like these, antibodies can be directly conjugated to agarose or magnetic beads with commercially...commercially available chemical agents. Directly conjugating the primary antibody has the additional benefit of...provides a large area for binding, but antibodies conjugated within the pores are often inaccessible to their... be much smaller than agarose beads. Proteins conjugate on the surface of magnetic beads, and are therefore...

... derivative. For propagating plasmids expressing the ccdB gene (important in Gateway cloning). F-mcrA ... derivative. For propagating plasmids expressing the ccdB gene (important in Gateway cloning). F- gyrA462...Chloramphenicol High competency cloning and propagation of large plasmids, ligated DNA, and libraries, nalidixic acid... and determine whether it is appropriate for propogating your plasmid or carrying out your experiment....experiment. History of E. coli strains E. coli are gram-negative, rod shaped bacteria that were named after Dr....Consequence dam DNA adenine methylase mutation (GATC) Preparing unmethylated DNA, important when trying...plasmid, encodes proteins needed for bacterial conjugation. Genes listed on F´ are wild-type unless indicated...

...antibodies must be either sourced already conjugated or conjugated in the lab. In the indirect approach, ...idea of the basics of immunoimaging. A scientist conjugates an antibody with a signaling molecule, the antibody... indirect approach, the signaling molecule is conjugated to the secondary antibody instead of the primary...of interest is found in low amounts or if the conjugated signaling molecule has a weak signal. For flow... are easy to source and are readily available conjugated to a number of different signaling molecules...., you can simply keep a stock of conjugated secondary antibodies on hand and use them to easily test different...sample. Incubate the sample with the primary (unconjugated) antibody. Perform a series of wash steps to...

...Ebola virus belongs to the group of nonsegmented negative sense RNA viruses, along with other viruses that...minigenome?   The EBOV genome is a nonsegmented, negative sense RNA strand approximately 19 kb in length...systems have been developed for many nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important...translated) by cellular machinery. NP encapsidates the negative sense, single-stranded RNA minigenome. The viral...polymerase to synthesize additional copies of the negative sense minigenome, thus modeling genome replication...transcription of the reporter gene. The polymerase will elongate the mRNA until it reaches the gene end signal,...precautions, these systems allow researchers to investigate viral processes in extreme detail. The mechanism...