...much as you can), form a peer mentoring group to get real feedback Write whenever you can, find supportive...Joanne's Reddit AMA Here are some concrete things you can do to develop those transferable skills while you...Demonstrating your ability to work well with others can be done in many ways… and many labs in academia benefit...advance science faster. Help other people when you can. The colleagues you help will be future advocates... team productive. Take all the opportunities you can to be communicating about science with others. If...organizations Another excellent perspective on this topic can be found on the UK Career Site for Researchers called...

...functional Cas9 molecule can cleave the target DNA. The inducible split cas9 approach can also be used for activation...Finally, the system can be adapted to cellular screening to identify molecules that can perturb protein transport...through Addgene so you can try these incredible new tools yourself! The tags can be found in the pBluescript...that this tool can also be used to visualize translocations, genome rearrangements and cancer-associated ...shoot us an e-mail at [email protected] and maybe we can write a blog post about it! Split Cas9 system The...expression. The plasmids associated with this article can be obtained through Addgene: Rapamycin-inducible...genes are turned on or off in a given area and the can do so in a very specific, precise manner. Previously...

... and leaky expression can occur in cells that don’t express Cre or Flp, what can we do about it?  Test...comprised of different sequences (loxp and lox2272) and can not recombine with each other. When Cre is introduced...either end of the transgene. Since loxp and lox2272 can not recombine, this ensures that the transgene is...They found that almost all off-target expression can be attributed to spontaneous recombination between...This indicates that the amount of leaky expression can largely be attributed to the level of spontaneous...vectors will have some level of recombination that can lead to leaky expression in cells that do not express...genomes contain a recombined/functional transgene that can be expressed in cells where Cre is absent. Additionally...

...many more. They can be targeted to the plasma membrane to measure membrane potential and can also be directed... detail below. Types of Voltage Indicators GEVIs can be divided up structurally into 2 main groups: voltage...separates the two components of the indicator, which can make the kinetics of voltage sensing to signal output...FRET Chemigenetic Sensors These types of sensors can have a variety of different components, but all contain...powerful for in vivo imaging especially since JFs can cross the blood brain barrier and many colors of ...expressed enzyme, typically on the cell surface, which can act on an upstream component, the chemical factor...the large number of sheer molecules and signal you can generate without disturbing the integrity of the ...

...and PI can provide feedback on your technical presentation, but those outside of your field can point ...Tailor your language to your audience so that they can digest what you’re saying, provide constructive criticism...determine how to tailor your presentation so that it can be understood by your audience. Use your poster as... aid, not a crutch. Even the most prepared of us can find ourselves talking to our posters, so if you ...aside to read through the list at the session so you can strategically use your time. Don’t have access to...yourself up for success. But sometimes, attendees can get sucked in to thirty-minute-long presentations...presentations, which constrains the number of posters you can see. If you are pressed for time, limit yourself to...

... animal facility which can be expensive to gain access to routinely, and it can be especially challenging...size, nanobodies can be synthetically expressed off of plasmids in bacteria, and they can target epitopes...10-30) of sybody candidates are sequenced for identification. All unique sybodies can be subcloned into...are small (12-15 kDa) single-domain proteins that can bind effectively to protein targets, similar to how...selection platform was designed so that any standard lab can quickly select for sybodies targeting a membrane ...regions that bind to the antigen. The Seeger lab can directly provide the three sybody libraries in the...contain serines and threonines at positions that can be randomized for creating the libraries (Zimmermann...

...of the amplicon. As the probes can have several different markers, it can also be used to run multiplex...gene exists, there must be assays that researchers can use to quantify said copies. They do so using copy...your sample relative to your normal control - you can set up your calculations so that your assay output...not particularly sensitive. The fluorescent signal can be affected by AT-rich DNA and the length of the ... as noted above.   In both applications, results can be affected by DNA quality and primer design, particularly... schematic representation of the Taqman reaction can be seen to the left, courtesy of UPODMG 1516 irivbrro...gene of interest and reference gene, copy number can be determined through concentration values, rather...

...Usage Universal. Can generate all adult cell types. Limited. Only specific cell types can be produced ...self-renewal rate and can be used universally to generate many adult cell types. Like ESCs, iPSCs can differentiate...journal. Stem cells are special types of cells that can develop or “differentiate” into more specialized ...ectoderm, and mesoderm. In essence, pluripotent cells can be thought of as descendants of totipotent cells....discuss in detail below [2]. Multipotency: When a cell can differentiate into multiple cell types of the same... As the name suggests, cells under this category can differentiate into only one cell type. Muscle satellite...cells found in the adult muscle are unipotent. They can differentiate only into myogenic precursor cells ...

...stability, GFP can be used in lineage tracking capacities in cell fate studies. It can also be used, when...are active. Further, GFP can label transgenically modified ES cells, which can then be used for implantation...become a mainstay in molecular biology. Scientists can easily utilize GFP-containing plasmids as a means...Fusion tagging: One of the most common uses, GFP can be fused to the N- or C-terminus of a protein, which... GFP under the control of a promoter of interest can be used to effectively monitor gene expression from...fluorescent biosensors here. Optogenetics: Scientists can use light to detect, measure, and control molecular...cells have successfully taken up the plasmid. This can serve as an alternative to antibiotic selection. ...

...Enzymatic reactions on tissues can be cumbersome Tyramide reagents can be expensive Can only stain with one HRP-RANbody...so only one HRP-conjugated RANbody can be used at a time. Samples can be sequentially stained with two ...the RANbody plasmids from Yamagata et al can be found here. Can’t find a RANbody against your favorite protein...because they are an animal derived product, there can be lot-to-lot variability in activity. No Genetic...which are discussed in more detail below. The HA tag can be stained for with an anti-HA antibody and serves...detecting GFP. Spaghetti Monster tagged RANbodies can be detected either with a labeled anti- HA or -MYC...on Addgene.org More nanobody expression plasmids can be found here Watch this video to learn more about...

...another protein — known as a reference antigen — that can also bind to that antibody. Then, the protein-antigen...the direct and indirect ELISA (more details below) can also be adapted to a competitive format. ...antibody. Next, its substrate is added and the reaction can be visualized via a colorimetric reaction. The direct...two different antibodies. As mentioned above, it can also be used to characterize antibodies against the...amplifies the signal as multiple secondary antibodies can bind to a single primary antibody. The secondary ...your target protein. The OD of experimental samples can then be plotted along this line to calculate the ...   The maximum OD that most microplate readers can detect is 4.0, so if you max out your standard curve...

...between these modules (linker length can and should be varied as it can have important implications for the...dCas9) to modify chromatin. A fluorescent protein can still be included for visualization purposes, but..., and the receiver cell turns green. This system can be used in many cell types including endothelial ...cell. Image from Tang et al., 2020.   G-baToN can also be applied to other fluorophore/antibody pairs...BFP-labeled receiver cells. What’s more, G-baToN can be used to bring cargo, such as protein or DNA, to... would also result in depletion of the FP, which can be easily visualized.  The tool-kit consists of highly...knock-in repair constructs. These repair constructs can be used to integrate the tagged AID* into genomic...

...aren’t just limited to large volume facilities; it can be useful in academic labs where postdocs, students...learned over the years with the hope that our insights can help steer you in the right direction when writing...biology is messy. Every single step in a lab procedure can and will fail. Some step failures require re-attempting...cases. The following code examples show how software can be structured to allow for the messiness of biology...problem? As we said above, any one of these steps can fail but, in our current setup, there are no explicit...dealing with the samples to newer lab members. We can set ourselves up for success by giving explicit instructions...data for lab procedures. With this data in hand, we can go back and optimize our procedure to make the whole...

... 5’ or 3’ sequence homology cannot be assembled using this method, but can be assembled with Golden Gate...overhangs are not part of the recognition sequence, they can be customized to direct assembly of DNA fragments...fragment with 5’ overhang TGGA and 3’ overhang TCCG can be ligated into a vector containing those overhangs...terms of hands-on time, as digestion and ligation can be done in one 30-minute reaction. The destination...cloning is its scalability. Unique 4 base overhangs can be used to assemble multiple fragments - up to 10... very small/very large fragments, these problems can be overcome by screening a higher number of potential...encoding eight amino acids, but Golden Gate assembly can be designed to be scarless. Golden Gate assembly ...

... you can download an annotated GenBank File or a SnapGene file for the sequences listed. You can then ...positions within the plasmid. The list of enzymes that can be detected is 5x greater than our previous mapping...cloning. With this more functional display, you can start thinking about your next cloning experiment...depositor provided a custom annotated GenBank file, this can be found on the main plasmid page under the "Resource...sequence For more immediate, high-level analysis, you can click directly on the “Analyze Sequence” button to..., but also have interactive functionalities. You can now hover over any feature, enzyme, primer, or ORF... product, and a description of its function. You can also click one location in the plasmid and then a...

...that can be harvested from the surrounding media. Nonetheless, there are a variety of ways you can optimize...increase the AAV concentration. In this flowchart, you can see the dwindling proportion of AAV particles that...their way to the nucleus where their gene cargoes can be expressed. The bottom line is that a lot of AAV...for this function is not yet fully understood, we can imagine that this transport is probably not 100% ...to drop off drastically with lower titers. As you can see in figure 2, the expression from the 5x1011 titer...considerations If you have high quality AAV, you can address the issue of low titer by concentrating your...AAV production. We’ve listed some simple steps you can take below but you should read our AAV Production...

...research practices (QRPs) can quickly lead us to find statistically significant results that are not robust...difficult to do?  There are four reasons that research can be difficult to replicate. Unclear methods Until ...false negatives in replications are unavoidable, but can be minimized with high powered research designs. ...population, new genetic variation, or different handling can all be important.  If we eliminate the problems above... out the same as an original research finding we can be more certain that some important, previously unknown...other materials. The use of reporting guidelines can also help remind us to include all of the essential...before conducting the experiment (note: registrations can be embargoed while the research is ongoing, but all...

...everything you do in the form of a protocol, and you can add in personalized steps like “check drawers for... next to each step as you complete it. Yes, this can be incredibly tedious, but it also works, so do it...organization of your workspace and your workflow can have a huge impact. Test different approaches, like... up your pipette tips to match your plate layout can help you track samples as you add them. Moving your...from your left side to your right as you use them can help you remember what's been added and what hasn't...hasn't. Developing simple habits like these can consistently reduce errors as you work, and the possibilities... If you need to focus, a large set of headphones can act as a Do Not Disturb sign, even if the headphones...

...occasional errors. That can be advantageous if you are trying to knock out a gene. One can also exploit homology-directed...people, we can use this kit to knock in those mutations. Once that's done in stem cells, we can differentiate...: For people unfamiliar with this new TALEN kit, can you describe it for us? Musunuru: TALENs are a type...type of genome editing tool with which one can introduce changes into the genomes of mammalian or other...is that the DNA-binding domain is modifiable and can be programmed to bind to essentially any DNA sequence...template for repair. By using these two scenarios, you can either knock out a gene or knock in a mutation. Addgene...more of an apples-to-oranges comparison. Addgene: Can you compare the TALEN kit to CRISPR/Cas9? When would...

...for viewing, editing or making plasmid maps, but can also analyze and annotate any DNA sequence. SnapGene... also has a suite of molecular biology tools and can make plasmid maps. Free for academic users. Serial...be “cleaned up” or changed in some way before you can analyze them. These tools help with that.  DNA sequence...Cloning ligation reaction calculators These links can help you calculate how much insert and backbone you...Calculator: This tool works for any cloning method. You can select from five different insert:backbone ratios...calculators Lab math calculators These calculators can help you with common calculations performed in the...iPhone Reagent and Data Repositories These links can help you find experimental and/or validation data...