...the primer only anneals once within the entire construct. Is the primer an appropriate distance from the...

...a plasmid to determine the size of the entire construct or excise some or all of an insert from it. Before...

...complicated procedures. Plasmid Cloning Protocols for constructing and analyzing your plasmids. Virus Protocols...

...Background Information The final step in the construction of a recombinant plasmid is connecting the insert...

...generate the four initial unique constructs and later combine all four constructs into one 15 kb insert. Table...Table 1: MXS Constructs for Labeling Cellular Structures Construct Fluorophore Excitation/emission ...used to incorporate each element into the final construct. This strategy can be used to build vectors with... used to produce complex fluorescent reporter constructs. These fluorescent reporters can be used to detect... combining these components, one can generate constructs to visualize cell cycle dynamics, titrate inducible...clever applications. In one example, modular constructs generated with MXS-chaining were used to label...structures in live cells. Four individual expression constructs were created, each containing a unique fluorescent...

...tRNA-gRNA constructs or a 1-4 base tail from the second tRNA in the tRNA-gRNA-tRNA constructs. gRNAs with... PTG constructs in rice protoplasts. Compared to matching traditional gRNA-expressing constructs, transcript...plants, comparing traditional gRNA constructs with PTGs. In PTG constructs containing two gRNAs, they found...with the use of standard single gRNA constructs. When PTG constructs with five gRNAs were tested, they found... weak, and low expression of gRNAs from these constructs could lower genome editing efficiency. One ...polycistronic transcript. Although this method shortens construct size, it requires coexpression of Csy4 with Cas9...indicates that gRNAs could be incorporated into these constructs without compromising cleavage efficiency. In ...

...Brainbow-2.1 construct can express one of four colors (n-GFP, RFP, YFP or M-CFP.) The construct contains ...the construct. Sanes and Lichtman used pronuclear injection to insert these Brainbow constructs into...distinguish individual cells. The Brainbow-1.0 construct contains three fluorescent proteins: RFP (red)...the same. The first fluorescent protein in the construct, OFP (orange), is expressed when Cre is absent... tandem integration of multiple copies of the construct (about 8 in Brainbow-1.0 mice.) The combinatorial...a rainbow of colors. With three copies of the construct, one would expect ten colors (see table to the... example, Brainbow-1.0 expresses RFP when the construct has not undergone recombination. This default ...

...generate negative control constructs — more on this later. For more on constructing fusion proteins, check...makes it easier to generate a large number of constructs for high-throughput screening of PPIs. For example...generally involves three steps: fusion protein construction, cellular expression, and quantification (Kerppola...described in more detail below: Fusion protein construction Fusing FP fragments to your PIPs requires careful... Connection. Cellular expression Whether BiFC constructs are expressed transiently by transfection or ... comparing the expression levels between BiFC constructs and their endogenous counterparts using western...blotting. The subcellular localization of BiFC constructs should also be compared against those of their...

... recognition sites do not appear in the final construct, allowing for precise, scarless cloning. The cloning...digestion/ligation and do not appear in the final construct. The destination vector contains sites with complementary...insert may spontaneously religate, this transient construct retains functional Type IIS sites and will be ...ligation product is irreversible because this construct does not retain the enzyme recognition sites. ... sites after ligation favors formation of the construct of interest. Although efficiency may decrease .... The popular Gateway cloning system produces constructs with an attB recombination scar encoding eight...IIS sites such that custom TAL arrays could be constructed quickly and efficiently in just a few steps. ...

...Expression of the optogenetic constructs Expression of your desired optogenetic construct in the brain region and...or camKIIα drive most of the constructs. As an alternative, some constructs have been engineered with a...promoter in the AAV construct. Stereotaxic surgery  As mentioned above, AAV opsin constructs are injected ... also generously made many of his optogenetic constructs available through the vector cores of various...Deisseroth lab homepage for details about types of constructs available, instructions for obtaining a material... University of North Carolina) that offer his constructs. Importantly, control viruses (not expressing... injection of an adeno-associated viral (AAV) construct using stereotaxic surgery (see videos below). ...

...into multi-TU constructs, known as multigene constructs. Construction of multigene constructs, through multi-TU...DNA parts, transcriptional units, and multigene constructs. Let’s start by examining individual parts and...Assembly of multiple Level 1 TUs into multigene constructs. Figure 2: The three assembly levels ...pathways, MoClo has been used to assemble multigene constructs capable of performing production and subsequent...prime choice for assembling an array of targeting constructs and delivering them to these organisms via another...system for standardized assembly of multigene constructs. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371...

...Assembly allows the production of scarless DNA constructs using homologous regions to guide the joining...its goal of creating a choice of standardized constructs and tools for the rational and simple shuffling... also allows the creation of standardized DNA constructs, but its use of Type IIS restriction enzymes ...mutations in one or multiple fragments, and generate constructs for producing single-guide RNAs – but it also...HiFi DNA Assembly Mix include:  Generation of constructs with multiple point mutations Cloning sgRNAs ...Production of Yeast Expression Cassettes Generating constructs with multiple point mutations NEBuilder HiFi ...thousands of base pairs. Cloning sgRNAs Generating constructs to produce sgRNA for CRISPR experiments can be...

... Addgene. Here, we take a closer look at the construction and validation of some of these tools created...collection.  Creating the Plasmids To begin constructing the plasmids for expression of mAbs, the Trimmer...each from a constitutively active CMV promoter. Constructs were then verified by both diagnostic restriction..., the next step would be to validate your new construct by Sanger sequence analysis. But for this research...correct clones, it was determined that for some constructs more than 90% of the colony PCR validated clones...result, subsequently cloned into the expression constructs. The Trimmer lab found that the recognition sequence... to greatly increase the number of functional constructs that were created. This greatly limited the number...

...-Gγ chimeras, and two untagged Gβ constructs. For the Gγ constructs, the GFP2 tag is fused to the N-terminus...TRUPATH BRET2 biosensor system was developed by constructing 14 luciferase donor Rluc8-Gα chimeras, four ...N-terminus of the protein. For the Gα constructs, localization of the Rluc8 was more challenging. The Roth... not possible to generate an optimal Rluc8-Gα construct for two of the 16 non-visual G protein transducers...Gα-Rluc8 plasmid, and the appropriate Gγ-GFP2 and Gβ constructs. Cells are then treated with the luminescent ...

...technique involves the introduction of a transgene construct containing fluorescent proteins (XFP) of different...expression of genes farther downstream in the construct. As RFP and OFP are directly downstream of the...downstream OFP, GFP, or RFP. Cells that express the construct but do not undergo recombination can be detected...of the fluorescent proteins from the injected construct. The fluorescent protein expressed in a single... recombination. In addition to the XFP in the construct, dBrainbow includes small epitopes fused to each...used in zebrafish by injecting a CMV-Brainbow construct at the embryonic stage. This technique could be...specific tissue. XFPs are introduced using a plasmid construct called Zebrabow-GateDest containing an RFP, CFP...

...plant-pathogen interactions, and her aim is to produce constructs and edit genomes so as to make plants, and agricultural...there are many, so if you want to make a 10-gene construct, you don’t have to worry about repeating elements...from the kit and getting started making your own constructs with Golden Gate MoClo. The barrier for those... for short projects and they end up making 30 constructs. They’ve never done any kind of cloning and they...tools with the plant community. We’d like to add constructs to allow people to, for example, knock out a ...complementation, with a knockout and expression construct on the same piece of DNA. We do a lot of protein-protein...system for standardized assembly of multigene constructs. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet...

... rely on the random integration of a reporter construct in close proximity to an endogenous gene. Each...detail below), but fundamentally the reporter construct monitors the expression of a nearby gene. The ...an uncharacterized gene. The actual reporter construct can be something phenotypic (such as a fluorescent...monitor. Gene traps Gene traps are promotor-less constructs that contain splice acceptors. They require insertion...type of trap, there are many ways to insert your construct to generate a collection of monitored genes and...

...lab, if you read a paper and saw an interesting construct you wished to use, you could either make it yourself...Addgene makes it so much easier to share your constructs from lab to lab. Promega supports the Addgene...only 19 kDa. An example is the CRISPaint-NLuc construct (Plasmid #67178) for use in the system outlined...fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed to be packaged into a retrovirus...reporter can improve deep tissue imaging. The constructs for GpNLuc and OgNLuc are available through Addgene...Antares as compared to Fluc in this model. The construct for Antares is available through Addgene (Cat....dynamics in cardiomyocytes. Enhanced Nano-Lantern constructs are available through Addgene: The Turquoise ...

...change gloves between constructs if you are transforming more than one construct at a time. For detailed...begins. If your plants begin to flower before your constructs are ready for transformation, you can cut off...lots of seeds. I typically use 8-10 plants per construct I transform. More seeds mean more chances to recover... over-expression and it is likely that such a construct will be silenced in subsequent generations. You...generation. You also have no control over where your construct will be inserted. If your insertion occurs in ...High-Performance Vectors for Creation of Fusion Constructs in Transgenic Analysis of Plants’, Bioscience...

... GFP. Once they’d found suitable pairs, they constructed three components: GBPa-VP16 (activation domain...domain); and a UAS-driven luciferase reporter construct. In the absence of GFP, no reporter output was... that YFP and CFP can activate certain T-DDOG constructs similarly to GFP, but commonly used red fluorescent.... electroporated T-DDOGs, GFP, and a reporter construct into the mouse retina. GFP expression robustly...of DsRed+ cells also expressed GFP. Using AAV constructs, Tang et al. found that Cre-DOG could be delivered...proteins. For proteins with a modular structure, constructing split variants should be relatively straightforward...