...these biosensors can be found at Addgene. Split FPs – some FPs (e.g. GFP, Venus) can be split into two...Photoactivatable / Photoconvertible: These proteins can switch their color when activated by a specific excitation...wavelength. This means that the emission wavelength can change. In a few cases, the initial state of the ...proteins change their color over time. Therefore, these can be used as “timers” for cellular processes following...they will form the full FP and fluoresce. Split FPs can be used to determine the proximity of two proteins.../em peak. Therefore, choose FPs that your system can excite, and detect the emission. For example, if ...yellow light). Note that some combinations of FPs can cause an effect called FRET (fluorescence [or Förster...

...backbones and specific primers you can use to confirm your clones. You can also find a list of popular sequencing...gRNA insert or not. A blue-white screen, however, can give you this information (more on this below). Plating.... lacZ encodes the enzyme 𝛽-galactosidase which can hydrolyse lactose. Fortunately, for the lab researcher... will just see the vector backbone band. Or, you can choose an enzyme that cuts once in the plasmid backbone...one band. These are only a few examples of how you can use restriction digest to screen your clones, but...using restriction digests to analyze plasmids, which can help you visualize this process, or visit our restriction...restriction analysis blog post. Colony PCR Colony PCR can detect the presence or absence of DNA using lysed...

...These whorls can be quantified for individual cells, and the number of 'normal' looking cells can be counted...that are engineered can be shared to simplify the comparison by others. Together, we can build a valuable...probes. You can follow him on twitter: @joachimgoedhart.   Marieke Mastop is a PhD candidate at the Section...tendency to form higher order aggregates. This property can be tolerated in some applications (e.g. labeling ...detail here. There are a variety of methods that can be used to measure your fluorescent protein’s propensity...proteins to form homodimers or higher order oligomers can be analyzed by several techniques. Gel filtration...spectroscopic methods (based on fluorescence polarization) can be used to detect homodimerization. However, all ...

... it can also detect expression of toxin mRNA from Clostridium difficile. As mRNA expression cannot be ...are doing. But aside from sequencing, scientists can also study the microbiome by using engineered genetic...IncP⍺-family-RP4 conjugation system. This system can deliver plasmids to both Gram-positive and Gram-negative...plasmids (pGT) with various replicative origins that can be used for broad or narrow range of microbial species...a desired genetic payload (ex: GFP). Thus, cells can be both sorted using FACS and isolated with antibiotic...Collins’s lab developed a paper-based method that can be used to detect microbes and biomarkers on paper...method is the RNA toehold switch, a RNA hairpin that can be designed to bind and detect nearly any RNA sequence...

...that antibodies can do! For instance, some Fc receptors, once bound to an antibody, can act as immune modulators...to bind. We also know that changing the Fc domain can impact an antibody's binding affinity (Janda et al... the different immune responses? We got you! You can also review antibodies in our Antibodies 101 animation...different receptors, but their downstream effects can be sorted into three main effector functions.  ...Antibody-dependent complement deposition (ADCD) Antibodies can bind to a pathogen and physically prevent it from...effector function known as neutralization. But they can also bind to a pathogen and mark it as a target for... cell. If the pathogen is a large parasite, ADCC can be induced through a very similar interaction between...

... insertion line can then be isolated and screened. Depending on the marker, traps can be used as high-...frame, in fact, they can be activated a considerable distance away from a gene which can make identification...expression levels (high/low) and patterns (tissue/timing) can be monitored under various conditions without necessarily...uncharacterized gene. The actual reporter construct can be something phenotypic (such as a fluorescent protein... result in lethality before all of its functions can be observed. Functionally redundant genes – multiple...multiple genes which function in overlapping roles can mask the depletion of one another. Under these circumstances...reporter gene flanked by a minimal promoter which can integrate proximal to an endogenous gene to turn ...

...In the event that genome sequencing can be done, this technique can also help pinpoint the genes within...Nebulization units, which can be purchased cheaply, are small plastic containers that can easily be attached ...are hard to find Sequencing and cloning phage DNA can be difficult, in part because phages frequently alter...approximately gene-sized) fragments so that these fragments can be cloned into E. coli and subsequent proteins screened...screened for cell-binding abilities (6). This can be accomplished using a restriction enzyme with a short...DNA with phi29 polymerase prior to fragmentation can greatly facilitate downstream manipulations. Phi29...Phi29 polymerase is a phage-derived polymerase that can perform whole genome amplification (by a mechanism...

...you can jump from one type of position to another throughout your career. Plus, just one role can encompass...  Where can you do science policy? Because the field of science policy is so diverse, you can do it in... of work can happen at the national level within the executive branch or in Congress, or can also happen... programs can connect you with alumni before and during your application process so you can learn more... about virtual meet ups and happy hours that you can attend to help build relationships.  Reach out to...differently depending on how and where you pursue it. It can manifest via research and data gathering, regulatory...as a profession. Figure 1: Science policy can lead you to many paths! For a more details, check...

...biological. Even the most high-fidelity polymerases can make mistakes and lead to the accumulation of errors...errors in DNA sequences. In research, these errors can affect experimental processes, results, and subsequent...bacteria — has many points at which small errors can be introduced. Eventually, these errors could cause...originally intended. Sharing unverified plasmids can lead to far reaching consequences, from wasting time... Figure 1. Potential points where DNA errors can be introduced, indicated by exclamation marks. Error...interested in requesting one of these plasmids, you can request that it be fully sequenced by Addgene’s QC... or annotated plasmid sequences. These sequences can also be confirmed or theoretical. No matter the level...

...incubation time can improve the cell density and therefore the plasmid yield. You can optimize your growth... strain Differences between E. coli host strains can impact plasmid yield. Check the bacterial strain ...encodes a thermostable periplasmic endonuclease that can sometimes co-purify with your plasmid DNA and lead...derivatives, release large amounts of carbohydrates that can inhibit the cell lysis process. To ensure that lysis...the volume Starting with a larger culture volume can improve plasmid yield. Our lab often doubles the ...more volume = more cells = more plasmid. Plasmids can vary in copy number, the number of plasmid copies...specified by a plasmid’s origin of replication, but can also be affected by the plasmid size, nature of the...

...beads are a mixture between synthetic beads that can bind to a conjugated antibody and beads that are ...and they are non-specific. The compensation beads can bind to any primary antibody you add. Single color...controls are used to help you set up your voltages and can also be helpful for compensation. You should have...see all of your positive and negative cells. This can be achieved by adjusting your voltage using your ...otherwise be difficult to separate out. In Figure 2, you can see that, without the FMO, it would be difficult ...Figure 2: An FMO gating example. The FMO sample (A) can be used while setting up your gating schematic to...population will look like for a particular marker, you can use the reference plots provided by the antibody ...

...consist of various immune cells, and can be used as a whole or can be used to isolate specific subsets ...antigen, triggering a humoral immune response. ADAs can inactivate the drug and increase drug clearance, ...silico and in vitro assessment of immunogenicity can help evaluate risk of an antibody.  In silico prediction...specific antigen, pre-existing conditions, etc. that can vary widely between recipients.   In vitro prediction...subsets like CD4+ or CD8+ T-cells or monocytes. PBMCs can be further differentiated into dentritic cells or ... et.al, 2018). Thus, microscopic in-vitro models can be built that contain various components of the immune...Treating these cells with the therapeutic antibody can help us assess the effects of the drug on the actual...

...for CRISPR experiments can be laborious, requiring multiple cloning steps. You can simplify this process...homology at, or close to, their ends. This homology can be contained within the individual fragments themselves...doesn’t necessarily have to be. Regions of homology can be introduced to fragments via PCR using primers ...is freely available online. Its simple interface can guide you through both experimental and primer design...constructs with multiple point mutations NEBuilder HiFi can simplify multi-position site-directed mutagenesis...containing the desired mutations. This same process can be used simultaneously for the generation of one ...Further, there is no limit to where the mutation can be placed. With other techniques, when mutations ...

... baculovirus can vary depending on the protocol used. Plaque assays are standard but can take seven to...Introducing the systems There are many ways that viruses can be produced in the lab. The first decision you must...cells that will be used to make the virus. Viruses can be produced in many different cell types, spanning...produced in bacteria cells. However, some viruses can be produced in multiple different cell types, including...your current methods, understanding the differences can help you make an informed decision. Mammalian cells... The viruses Lentiviruses, AAV, and adenoviruses can all be produced in mammalian cells. However, it is...it requires transduction of more cells, while AAV can be titrated following purification using qPCR or ...

...for energy between neighboring fluorophores, FRET can be used to detect the inter- and intramolecular interactions...underlie protein function. However, FRET experiments can be difficult to implement in practice because FRET...complete FP results in a fluorescent signal that can be detected by fluorescence microscopy. Figure...the overexpression of fusion proteins it requires can lead to aberrant subcellular localization of the ...genetically tractable organisms lags behind. BiFC can also be used to screen for small-molecule inhibitors...fragments. In the absence of this information, you can fuse each FP fragment to the N- and C-terminal end...case, overexpression should be avoided because it can generate high background fluorescence levels due ...

...takes half a year, and generating a transgenic plant can take even longer, a transient plant leaf transformation...methods. Infection with Agrobacterium rhizogenes can be used to develop stable hairy root lines in tissue...investigating gene function, as these experiments can be done in days to weeks instead of months to years...transferred into the plant. In the laboratory we can utilize this natural process by replacing the genes...gene so that the transformation and gene expression can be tracked. During a transient leaf transformation...establishing a new transformation protocol as it can easily show which tissue have been transformed. But... Agrobacterium lacking the GUS gene since plants can have endogenous GUS-like activity (Hu et al., 1990...

...stain from the background. Dyes that can pass through cell membranes can prove useful for live-cell imaging..., such as proteins, nucleic acids, or glycans. Fluorophores can also be covalently attached to self-labeling...have been developed throughout the past century, it can be overwhelming to select the optimal dye or fluorescent... genetically encoded tag is required. These dyes can also be attached to an antibody to detect various...2022). These variations directly impact the way you can utilize these dyes. The most-used fluorescent dye...organelles like mitochondria, and different dyes can result in varying cytoplasmic background signals ...fluorescence of fluorescein isothiocyanate (FITC) can be stably combined with nanoparticles, maintaining...

... all lightsheet microscopes can cover samples of this size, those that can will completely image large...insights, it can be overwhelming to a novice microscopist. Hopefully this brief synopsis can help point ...addition to this, the use of multiple fluorophores can add a second layer of contrast, color contrast, to...Disk) increases the speed at which a confocal image can be acquired. Assembling an image pixel by pixel is...than one hole is illuminated at a time, the image can be acquired more quickly. 2-Photon Microscopy 2-Photon...of blue. Only at the focal point of the objective can a a high enough density of red-photons be establish...light is entering your hand, only orange/red light can be seen exiting through the tissue. Therefore, 2-...

...is covalent, so free dye can be washed away. In addition, labeling contrast can be improved using disulfide-containing...reagents can bind numerous endogenous biomolecules in cells nonspecifically, and they cannot cross cell...the first fluorophore (the donor) is excited, it can transfer energy directly via resonance to the second...30-100 Å, depending on the probes). In fact, FRET can be used to derive a precise physical distance between... fluorophore labeled and that these fluorophores can undergo FRET when they are near each other. How do...biomolecules each containing multiple binding crevices that can nonspecifically soak up your fluorophore package....delivered to the wrong address in a different city, you can still develop a successful FRET-based assay since...

...plasmids are more stable, can be easily cloned in different bacteria, and can be transferred to various...broad host range plasmids can be used to create recombinant vectors that can replicate in different bacteria...recombinant vectors can be modified further to increase the range of hosts in which proteins can be expressed...natural plasmids and how they function in nature can inform us on how to create better recombinant vectors...in plasmids in nature, and provide tips on how we can harness those features to create broad host range...determined by the different hosts that a plasmid can transfer to, replicate in, and persist in. Some plasmids... range, while others have a broad host range and can replicate and stably maintain the genes they carry...