We narrowed to 513 results for: gats

...on the Multisite Gateway® technology from Life Technologies. The MultiSite Gateway Kit is a customizable... systems. The MultiSite Gateway Kit contains a set of 12 promoter-less Gateway® entry clones to be used... phage integrase system which is the basis for Gateway cloning. The combination of all 11 att pairs in...site and a linker region. The PCR product is then ligated into the MCS/SacI site in pUCXKT, removing the ...customizable Gateway®-based toolkit that allows scientists to generate fusion proteins from any ORF available...as a standard entry clone. ORFs from an ORFeome Gateway library or any PCR-amplified ORF cloned into pDONR221...heterologous promoter outside of the Multisite Gateway® cassette, you can specifically express your new...

...pCAGGS-TAL-NC2 Charles Gersbach pcDNA3.1-GoldenGate, pcDNA3.1-GoldenGate-VP64 Maria-Elena Torres-Padilla pTALYM3...74334 pCR8-Magneto2.0-p2A-mCherry Gateway 74333 pCR8-Magneto2.0 Gateway 74307 pAAV-CMV-DIO-Magneto2.0...PRESTO-TANGO kit which allows researchers to interrogate the druggable human GPCR-ome using a quick, affordable...destination vectors using low-cost, one-pot golden gate cloning that results in the creation of many E. ...The collection is compatible with both standard Gateway cloning and the FNLCR Combinatorial Cloning Platform...Red recombinase enzymes, as well as a dominant-negative mutator allele of the E. coli MMR protein MutL...Ekker’s lab, is a modified version of the Golden Gate TALEN kit (GGT) that streamlines the assembly of...

...experiment-based investigation of a hypothesis: proper controls! For every experiment, an investigator needs a ...empirically.  Negative controls: untreated cells, empty vector control, and non-targeting control Negative control...plasmid-based experiments employ transfection, negative, positive, and replicate controls.  Transfection...mentioned above) could also serve as an important negative control. In this context, the empty vector control...your observed result. Non-targeting controls are negative controls that produce a similar product, but do...be highly specific to the experiment and the interrogation of the independent variable. Replicate controls...experiment, incorporating additional positive and negative controls, performing multiple replicates, and ...

...valuable when first optimizing conjugation in strains that don’t conjugate that well. This is because in...exchange. Here, a mutated allele introduced from a conjugative plasmid replaces the chromosomal copy. This is...bacteriophage lambda pir protein. This allows conjugation between a pir-containing donor and the recipient...inspired by this work and sought to develop these negative selection markers for allelic exchange. To do ... which we call “pTOX” in a broad group of Gram-negative Enterobacteriaceae including in E. coli O157:H7... to facilitate modification of strains where conjugation may be difficult, we have also included versions...in these circumstances, the conjugation can be so inefficient, that instead of the resulting colonies ...

...than the negative control, and a weak signal has a longer exposure time closer to the negative control....specificity and exposure time. We first have a negative control which does not have primary and only contains...autofluorescence i.e., background noise. We then compare the negative control to a sample with the primary included ...background noise. If the sample images similarly to the negative control, that informs us that the primary antibody...non-specific binding. If the sample differs from the negative control, such as showing a very bright signal ...exposure time of the primary antibody compared to negative control also determines the signal’s strength....comparing your sample to. For example, we use our negative control to compare exposure times of our samples...

... the negative selection marker, but cells with random recombination may incorporate the negative selection...One of the most powerful strategies to investigate a gene's function is to inactivate, or "knockout",...resistance gene (NeoR) flanked by homology arms. The negative selection marker HSV-tk is used to select against...construct has randomly integrated into the genome, a negative selection marker like the HSV thymidine kinase...resistance gene and then a counter selection for the negative selection marker - this latter step will kill ...thus disrupted, or knocked out. Use positive and negative selection to find correctly recombined cells. ...selection marker selects for recombination, the negative selection marker selects against improper, random...

...overwhelmed if you don’t delegate - as I quickly learned! I was a poor delegator at first. I knew what ...team - which is every manager’s dream. Learn to delegate effectively  As a manager your role is not to ...more work (this is a big hint that you are not delegating enough!). There is no way around the fact that...possible so I could more easily identify what to delegate and to whom (make sure that person has time for...available, and encourage questions. Unless you’re delegating a recurring task to a seasoned report, there ...there should be questions! Remember that delegating is not just a way to create more time for yourself, but...need to check myself sometimes to make sure I am delegating well, but it does get easier over time! Communicate...

...introduce Mobile-CRISPRi by bacterial conjugation For gram negative bacteria, Mobile-CRISPRi is introduced...Mobile-CRISPRi stably integrates into the genome and is propagated during normal DNA replication and cell division...introduced using the bacterial integrative and conjugative elements in Bacillus subtilis (ICEBs1) system...system. ICEBs1 carries the genes needed for conjugation, but can also carry cargo genes, like Mobile-CRISPRi...bacterium of interest following IPTG induction of conjugation genes and bi-parental mating. Following excision...genome, the Mobile-CRISPRi ICE element forms a conjugative plasmid-like DNA circle and is transferred to...et al. "Biology of ICEBs1, an integrative and conjugative element in Bacillus subtilis." Plasmid 86 (2016...

...pSC101 through a negative feedback cycle. As with DnaA, high concentrations of RepA negatively regulate its... require the host's replication machinery for elongation and termination. These types of plasmids tend...is carefully regulated by a mix of positive and negative regulators. The genome of E. coli is about 4.6...opposite directions away from the ori. In the elongation phase, a DNA polymerase synthesizes a new DNA... best tools to regulate DNA replication. DnaA negatively regulates its own expression, so high levels ...copy number for a given plasmid has been reached, negative feedback circuits reduce replication of the plasmid...proteins. In nature, such plasmids ensure their propagation by limiting their copy number and compensating...

...generated against a specific antigen and then conjugated with a fluorescent molecule allowing one to “...secondary antibody that targets the primary is conjugated to a fluorophore (Figure 1).   Figure ...you can begin to see why generating fluorophore-conjugated versions for each primary antibody could get ...secondary antibodies that target common IgGs and then conjugate them with an array of fluorophores. It is common...then use the corresponding secondary antibodies conjugated to distinct fluorophores to visualize multiple...apply, which binds to the first antibody and is conjugated to a fluorophore. You may need to optimize the...or have been manipulated to overexpress it. For negative controls, use samples in which the target is known...

... is directly conjugated to a detection substrate, such as a fluorophore, or a conjugated secondary antibody...a protein in a tissue section then a directly-conjugated antibody will provide better resolution as these...immune complexes are smaller than a primary:secondaries:conjugates complex. If you are trying to visualize... Some applications use either direct (primary-conjugated) or indirect (secondary) methods. In those cases...accomplished by using a panel of primary antibodies conjugated to different fluorophores or a panel of primaries...species, then you can choose different fluorophore-conjugated secondary antibodies specific to each isotype...troubleshoot and be sure to include proper positive and negative controls as you get your assays up and running...

...-RP4 conjugation system. This system can deliver plasmids to both Gram-positive and Gram-negative cells...Metagenomic Alteration of Gut microbiome by In situ Conjugation (MAGIC). With MAGIC, scientists introduce an ...phyla that have taken up the plasmid through conjugation in vivo studies. However, they only identified... of modular parts that are assembled with Golden Gate Assembly. They built this toolkit upon the Yeast...retains the oriT for delivery into recipients by conjugation. The labs screened for the host-range of the ...fluorescent proteins into one bright particle that segregates into one cell during cell division. To achieve...PMC5963681.  Meyer, Richard. "Replication and conjugative mobilization of broad host-range IncQ plasmids...

...Addgene: And the BIL gates collection? Endy: The Boolean Integrase Logic (BIL) gates collection of parts...switches that enable cellular computing. These logic gates built from transcriptors are available at Addgene...Addgene spoke with Endy more about the BIOFAB and BIL gates plasmid kits, the Biobricks Public Agreement, and...bioeconomy. Addgene: What do these BIOFAB and BIL gates kits – the first to be made available via Addgene...systems, plumbing, computers, and now through BIL gates we've implemented them in DNA. We thought these ...work, watch Endy's 10-minute YouTube primer on BIL gates.) Addgene: Why is it so important to you that these... Jerome Bonnet et al. "Amplifying genetic logic gates." Science. 340, 599-603 (3 May 2013). Vivek Mutalik...

...or the primer design. Use a negative control strain. A good negative control strain is an untransformed...with a positive clone amplifying a product and a negative clone resulting in no product. Additionally, this...clone will produce a larger size product then a negative clone without the insert. This type of primer ...on the number of background colonies on your no ligation control plate. The more background, the more colonies... positive clones (+) will give a band, while a negative clone (-) will not. Backbone-specific primers ...sized products for positive clones (+) compared to negative clones. Finally, orientation-specific primers ...important for insert-specific primers. If your negative control amplifies a product of the expected size...

...transcription. The HIS3 reporter system (e.g. the Gateway-compatible bait Y2H plasmid pEZY202  from Yu-Zhu...screens are prone to both false positive and false negatives, these experiments must be set up, conducted and...leads with other methods. More common are false negatives; up to 75% of known interactions can be missed...partners with other techniques! Minimizing false negatives: Interactions leading to detectable reporter signals...parameters can lead to a high number of false negatives in Y2H screens. Testing your reporter system ...protein doesn’t function in yeast! Many false negatives stem from or are exacerbated by expression of ...expression levels and systems can alleviate false negatives that arise from low or faulty expression of your...

... the same name. The tag has a net negative charge with 4 negatively charged and 1 positively charged amino...hemagglutinin. The tag has two negatively charged amino acids for an overall negative charge. This tag has been...common tags, FLAG is the most charged, with 5 negatively charged and 2 positively charged amino acids....localization and solubility, so be on the lookout for aggregation of your protein of interest!   Ready to start...Arribere, J. A., et al., Translation readthrough mitigation. Nature. (2016) 534, 719-723. DOI: 10.1038/nature18308...

...outside their field, the answers were primarily negative. Stupid, overwhelmed, discouraged, tired…words...from AAV Purification by Iodixanol Gradient Ultracentrifugation First, you’ll want to identify the reader...for AAV Purification by Iodixanol Gradient Ultracentrifugation. The reader may be, for example, a graduate...between a gradient tube before and after ultracentrifugation so that a scientist can extract virus from...interfaces between layers are blurred after centrifugation and the syringe needle is inserted bevel-up...profile photo of an iodixanol gradient after ultracentrifugation. The interfaces between layers are muddled...illustrates a gradient tube before and after ultracentrifugation. The viral supernatant migrates through increasingly...

...detected using an unconjugated primary antibody, then visualized using a fluorophore-conjugated secondary antibody...Antibodies - typically, primary antibodies that are conjugated to a fluorophore, to avoid the use of secondary...the signal comes from the antibody-fluorophore conjugate, and is not expressed by the cell itself, there...lot of flexibility in which proteins you can investigate in each panel. An advantage of flow cytometry.... These could be visualized using fluorophore-conjugated antibodies (i.e., immunofluorescence), or by .... These applications are typically used for investigating signaling pathways or developing therapeutics...therapeutics. Experimentally, antibodies conjugated to fluorescent tags or other markers can be injected into an...

...Method Choose from: Gateway cloning, gene synthesis, Gibson cloning, ligation-independent cloning, restriction...Please indicate which growth strain Addgene should propagate your plasmids in: DH5α, NEB Stable, ccdB Survival...Survival, Pir1, or other. Whenever possible, we propagate plasmids in the standard cloning strain DH5α. For...For plasmids containing the ccdB gene, such as Gateway vectors, choose the ccdB Survival strain. For plasmids...choose the Pir1 strain. If your plasmid cannot be propagated in any of these standard strains, choose other...Distribute pending QC. The name of the Principal Investigator and Organization where your plasmids were first...

... antibody is also conjugated to another molecule, such as biotin. Next, a conjugated substrate is added... biotin-conjugated, streptavidin (which has a high affinity for binding biotin), is conjugated to the ...protein of interest is then detected by an enzyme-conjugated antibody. Next, its substrate is added and the...instead of detecting your protein using an enzyme-conjugated antibody, a primary-secondary pair is used. This...single primary antibody. The secondary antibody is conjugated to an enzyme for visualization. The indirect ...