... silencing mechanism. It is a useful tool to investigate the roles of specific genes or to knockdown genes...

...get started with AVEXIS, including positive and negative controls Zebrafish receptors Zebrafish antibody...

...pEZYshuttle Shuttle Modified pShuttle-CMV plasmid with Gateway cassette; See article for additional plasmids Zhang...

...disodium salt from a commercial supplier and conjugating it to the label of their choice according to ...

...cancers are caused by inactivating mutations in negative regulators of mTORC1. The hyperactivation of mTORC1...

...such as adenovirus or herpes virus, in order to propagate. This is due to the reliance of the AAV on certain...

...amplification Viral vector production Density centrifugation purification Addgene’s comprehensive quality...

...silencing rescue in plants - Martin Parniske. GreenGate Cloning System to create plant expression vectors...

... Cas9 Lentivirus Cas9 viruses can be used to interrogate the genome in a variety of ways. Cas9 proteins...

...identified by enChIP-MS - A putative role of HDX as a negative regulator. Hamidian A, Vaapil M, von Stedingk ...

...vectors from the Esposito Lab for use with the Gateway® Cloning Platform (Life Technologies™) to permit...

...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...