...can be used to assemble multiple fragments - up to 10 fragments are commonly assembled in a single reaction... Gibson assembly require 20-40 bp of homology at the ends of DNA fragments to specify assembly order, ...sequence homology cannot be assembled using this method, but can be assembled with Golden Gate. The popular...acids, but Golden Gate assembly can be designed to be scarless. Golden Gate assembly is also less expensive...recognition sequence, they can be customized to direct assembly of DNA fragments. When designed correctly, the...sites with complementary overhangs that direct assembly of the final ligation product. As shown below,...higher number of potential clones. Golden Gate assembly has a few advantages over other cloning methods...

...organization scheme for multi-part DNA assembly. Simply put, it assembles individual DNA components into larger...MoClo-compatible vectors. Level 1: Assembly of multiple Level 0 parts into TUs. Level 2: Assembly of multiple Level 1...IIS restriction enzyme and a ligase are used to assemble multiple DNA fragments together in a one-tube ... Fig 1. MoClo enables individual parts to be assembled into larger functional units which are transcribed... Understanding how individual parts can be assembled into TUs is key to understanding MoClo. By a similar...Construction of multigene constructs, through multi-TU assembly,happens via three steps: Level 0: Cloning of ...multigene constructs. Figure 2: The three assembly levels of MoClo. Image courtesy of Rob Hurt. ...

...modules. In the first assembly step, compatible Level 0 vectors are directionally assembled into a Level 1 vector...of Level 1 assembly if necessary. Combining multiple Level 2 vectors in the final assembly step permits...structural and spectral properties of two of the family members – one with an unprecedentedly-high brightness, ...relies on several observations: Rapamycin is a membrane-permeable pharmacological agent that can rapidly...extraneous proteins bound to its outer mitochondrial membrane. Sequestration is accomplished by knocking down...protein with a FKBP domain and a mitochondrial outer membrane protein with a FRB domain (MitoTRAP). Rapamycin...., J Cell Sci 2013 May 1;126(Pt 9):2102-13. Assemble multiple component plasmids from building blocks...

... cells in an organism, including the embryonic and extraembryonic cell types. In mammalian development...generation does not require embryos, their use skirts issues surrounding the use of embryos in research. With all...Beckmann, and W. Wurfel, Non-embryo-destructive Extraction of Pluripotent Embryonic Stem Cells: Implications...-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic...totipotent cells. An example of pluripotent cells is embryonic stem cells (ESCs), which I discuss in detail below...into two broad types according to their source: Embryonic stem cells ESCs are pluripotent stem cells that... abnormal and/or supernumerary preimplantation embryos that are produced during in vitro fertilization...

... PI helps lab members determine project "ownership" and author order PI lets lab members fight it out ...long list of written questions for the PI and lab members.  Make coffee or beer dates to talk to as many ... informative way of gathering data. Former lab members can give open, revealing information about the ...and lab head. While it’s good to talk to new lab members too, they may not have had experience writing, ...career opportunities with the lab head. Former lab members can give you a fuller picture. Consider talking...try to attend a lab meeting. Observe how the lab members treat one another (especially those low on the ...will work for you, ask questions like: Do lab members feel there are enough projects to go around? What...

...zebrafish embryo develops outside of the mother, making them ideal for studying vertebrate embryogenesis. To...investigations into environmental pollutants and health, embryo growth, brain function, and disease development...illustrate some of the reasons that zebrafish have been embraced as a model organism, I’ll outline a few of the...quality genome sequence A high-quality sequence assembly of the zebrafish genome was published in 2013,... when and where genes are active in developing embryos are available. Additional analysis tools have been...head, eyes, and tail. Compared to the opaque mouse embryo, where similar processes occur over days inside...the zebrafish is an ideal system to observe embryogenesis live. The origins, divisions, and movements ...

...method. Membranes The two membrane options are PVDF and nitrocellulose. PVDF is a sturdy membrane that can...low-background PVDF membrane, it also has higher background noise than nitrocellulose membranes. Pro tip! You...visualize proteins on a PVDF membrane without staining; simply wet your membrane with 20% methanol and place...designing the experiment. What detection method? What membrane? What should you block with? It can be so overwhelming... location of your protein. Proteins located in membrane-bound subcompartments, like the mitochondria or... protein is difficult to solubilize, such as a membrane-bound protein. There are many different lysis ...the transfer: you’ll need to pre-wet most PVDF membranes before the transfer and allow them to dry afterwards...

... Team, but I’m also an emeritus member of the Scientist Team, a member-by-association of the Development...at Addgene, an unofficial title that I happily embrace (and admittedly, I may have started that nickname...update how we make and distribute kits, I helped assemble a crack team of Addgenies that includes: Michelle...control efforts with depositors; Gizela, the lab member who knows how the physical kits are made and shipped...months, or even a few weeks. The important thing to remember is that every team should have a goal, or at the...and providing technical customer service. Team members are highly trained, and though there are necessary...particular function. Project team A Project Team is assembled to complete a series of tasks with precisely defined...

...and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol in cell membranes and...can lose membrane-associated proteins, but it is better at permeabilizing organellar membranes. Created... work is to try it. As you embark on your next IF adventure, just remember that even though these early...proteins that need a little help crossing cell membranes as they are not able to diffuse across natively...Permeabilization essentially punches holes in the cell membranes of your sample to allow antibodies better access...target is already on the extracellular side of the membrane, then this step may not be necessary. In fact,...and leaves many membrane-associated proteins in place. In contrast, Triton X-100 and Tween-20 are examples...

...-GfaABC1D-mCherry-hPMCA2w/b Expresses a plasma membrane calcium pump hPMCA2w/b Baljit Khakh 112000-AAV1...

...Immobilizing proteins on a membrane Once separated, the proteins are immobilized onto a membrane, usually composed...sticky areas on the membrane will bind to the proteins in the buffer, blocking the membrane  from binding to...are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using antibodies.   ...protein is separated in a gel, transferred to a membrane, stained with antibodies, and finally visualized... and high binding capacity. In addition, PVDF membranes can be repeatedly stripped of an antibody and ...follow the same general principle where the gel and membrane are “sandwiched” together with blotting paper ...proteins to migrate out of the gel and attach to the membrane.  In a wet transfer system the sandwich is completely...

...2008 Gibson Cloning Gibson assembly method Single reaction, can assemble large DNA sequences, no restriction...derived from the tyrosine-protein kinase Lyn) Membranes 3 mCherry 587nm/ 610 nm human β-Actin Actin ...same multiple cloning site (MCS). Modules are assembled one at a time by standard restriction enzyme digestion...upon ligation. As a result, once two modules are assembled next to each other, a third module cannot be placed...high-throughput cloning strategies available for assembling these modular types of constructs, and different... Golden Gate Type II restriction enzymes Can assemble up to 10 modules in a single step. Through iteration...restriction enzyme-based). Well suited for the modular assembly of highly similar or repetitive sequences. Intermediate...

...introduced a new assembly cloning platform called GMAP (Gibson assembly-based modular assembly platform). GMAP...Plasmids from:  March (Q1) | June (Q2) | September 2016 (Q3) | December 2016 (Q3) MARCH 2016Enhancer activity...GMAP is based on the Gibson Assembly method, which allows for easy assembly of DNA fragments by utilizing...streamlines the assembly of TALE repeats into a single-tube, 3 day process by utilizing pre-assembled trimers ...literature searching, DNA synthesis, and plasmid assembly. Luckily, researchers from Douglas Densmore’s ...Genomics. 2015. PubMed PMID: 26165496 GMAP - A new assembly platform for generating both simple and complex...overlaps). To expand the capabilities of Gibson assembly and make a more modular platform, the Jacks lab...

...only a small proportion were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic...DREADD receptors need to be located on the plasma membrane to be activated by the DREADD actuator after it.... Although we found a larger proportion on the membrane in mice neurons, most of them were still cytoplasmic...limited expression of hM4Di‐mCherry at the plasma membrane in monkey neurons, we next hypothesized that a...improve the transport of this DREADD to the plasma membrane. The haemagglutinin (HA) tag is a small 9 amino...resulted in abundant expression of hM4Di in plasma membranes in both species (bottom panel in Fig. 1). Our ...transgene of interest and its localization in the membrane. The lesson we learned from these studies is that...

...on the surface, giving the embryo a “spiny and prickly look” that resembled a hedgehog (Nusslein-Volhard...eyes. Scientist thought these thick “eyebrows” resembled those of the comedian Groucho Marx, so the gene...Institute! Sonic Hedgehog, an important regulator of embryonic development The largest conference room at Addgene...hedgehog exhibit abnormal segmentation during embryogenesis. Typically, regularly spaced denticles (bristles...bristles) outline the segments of the Drosophila embryo. In flies with non-functional hedgehog, the denticles...gradient exerting a different effect on the developing embryo depending on its concentration. Genetic mutations...mutant fly ovaries had “squiggly” actin fibers that resemble spaghetti. We have two plasmids in our inventory...

... option: Fragmid. Fragmid is a modular vector assembly process designed for a quick, plug-and-play approach...depositor John Doench reports even the greenest members of his lab, high school interns, were able to use...five days. Did you say… easier? Fragmid’s modular assembly structure takes a lot of the design work out of...  Once you have the components you want, simply assemble them into a destination vector via Golden Gate...Whatever individual components you choose will assemble in the expected module order. (It’s also why you...the Fragmid system by sequencing 60 individual assemblies created over five months. When they tested those...were perfectly correct. Because they created the assemblies in duplicate, they were able to confirm that ...

...uninteresting, the panel members all say the same thing and one panel member dominates the discussion?... no time for audience questions and an audience member asks a 5 minute question that is really a speech... a fun discussion. People are much better at remembering these things on a relaxed phone call than when...moving and to prevent any one panelist or audience member from taking over. My own personal rule is that ... get things started. Give a couple of audience members a chance to participate. For example, at a recent...nonprofit science organizations, I asked audience members to stand and describe unique nonprofit models that...in the audience. I always try to meet audience members before a panel discussion and often incorporate...

...Modular cloning is a popular DNA assembly tool used to build single- and multi-gene constructs for a ...is. MoClo background MoClo is a clever way to assemble several DNA parts in a specific orientation using...are reusable, saving time and money on future assemblies. If you are new to MoClo, give our MoClo 101 ...Kit has a wide array of DNA parts that can be assembled into individual TUs or very large (up to 20!) ...CIDAR offers a one-pot reaction wherein multi-part assembly can occur. It is quite extensive with a supplemental... most intuitive CRISPR MoClo application is to assemble multiple unique sgRNAs into a multi-TU targeting...save time and money on studies that require many assemblies or that compare the effects of different DNA ...

...diffuse through the cell membrane freely, while others require a permeabilized membrane to enter cells. Depending...Depending on the application, they can be membrane-permeant or membrane-impermeant, and the cells of interest...viability dyes are membrane-impermeant and can only enter cells with compromised plasma membranes — i.e., dead... The targeted epitopes can be motifs within transmembrane proteins, such as receptors, or post-translational... marker of interest is not located on the cell membrane but inside the cell. Due to their chemical nature...nature, antibodies cannot penetrate the cell membrane; hence, intracellular labeling with antibodies requires...Permeabilization, as the name suggests, perforates the cell membrane to allow for antibodies to enter the cell (Figure...

... would be injected into developing mouse embryos, the embryos allowed to develop into chimeric mice (with...would first introduce a mutation into a mouse embryonic stem cell (ESC) line, enrich and select for cells...fascinated by CRISPR and rushed to test it. I still remember the day, March 16, 2014, that I got my first preliminary...Streptococcus aureus, is as effective at editing mouse embryos as Cas9 from S. pyogenes and has the advantage ...of fertilized mouse eggs. Or, to avoid handling embryos ex vivo, the components can be electroporated into...Doench’s gRNA design blog post. Finally, always remember that you are working with RNAs, which are prone...payload! Last but not least, when using CRISPR, remember to appreciate the fact that it was first discovered...