...condition 1” points (POI and loading control) are smaller than the analogous green “condition 2” points by the...blot.   Total protein measurements as loading controls Total protein measurements are an accurate alternative... example gel (left) with bands corresponding to points on the plot of protein abundance versus band intensity...the exact same value. For the two solid POI points, this difference is reflected on the band intensity...intensity axis. But the loading control points are outside of the linear detection range. As a result, they have...Outside the linear detection range, band intensity measurements become extremely susceptible to error, since...for your POI. Figure 6: Total protein measurements are taken on the same gel or membrane (depending...

...all the variant PUF segments required to bind any 8 base RNA sequence. The segments are provided in Gateway... have any questions, please write to us in the comments section.  FX cloning kit This kit is designed...Translation of the standardized leader peptide prevents secondary structures in the mRNA encoding your...expression systems; the Cell-free expression test kit represents a set of 88 clones in a pCellFree vector that...quantitatively determine allele frequencies across timepoints or populations. This kit consists of a 48 plasmid...ordered progression to resistance." Antimicrobial agents and chemotherapy 57.11 (2013): 5373-5383. Walkiewicz...Assembly Kit PUF proteins are composed of repeat segments that can be engineered to bind specific RNA bases...

...fluorescent proteins, with recombination events specified by three loxP variants, but the principles remain the ...recombination to generate additional recombination events, and the synthesis of alternative loxP sites to...expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate recombination...either deletion removes one of the other loxP site variants, preventing further recombination. Brainbow-1.1...) The construct contains two tandem invertible segments, each with two fluorescent protein coding sequences...combinatorial effect of multiple, independent recombination events leads to a rainbow of colors. With three copies...Variations Between Brainbow Systems   Brainbow variants and applications beyond the mouse brain In addition...

...same output: a spreadsheet of read counts. You can use these counts to confirm the representation of your...when you need to amplify a library for your own experiments. Library amplification 101 Library amplification... lots of library DNA is needed for screening experiments, so the scale of the DNA prep is larger. And ...is similar for most libraries. Here are the key points you’ll need to remember when undertaking library...enough DNA and competent bacteria to ensure all components of the library are represented. Typically, Addgene...need to count how many times each of the library components is found in the read list. If you also sequenced... your library. When plotted as a histogram, the counts will give you a bell curve of the abundance of ...

...undervalued.” Connie Cepko points out the limited choices in hiring graduate students and postdocs when starting...be half of my day sometimes.” He points out that the copious amounts of email are probably because a PI... than just experiments and grant writing. You have to enjoy the mentoring role.” She points out that new... having projects ambitious enough that everyone wants to recruit rather than repel and hoard.” If you'...it is once it gets fully up to speed.” He also comments that it is also surprising “ how easily people...last post in this series “Fun in the lab!”   Acknowledgments Thank you to Dr. Tom Ellis (Imperial College...

... interested in using GCE, here are the basic requirements of use. First, and most obviously, you need ...incorporated ncAA, ready for you do to any number of experiments with. The catch The rapid expansion of GCE led...That many GCE technologies have idiosyncrasies presents a significant barrier for those wishing to use...GCE aren’t able to successfully complete their experiments and many others are dubious enough that they ...optimization parameters the same for all three components, meaning that the final protocol may involve ...’s not uncommon to find protocols that require reagents which aren’t broadly or easily available, so you...protocol that you’ll have to rework to include reagents accessible to your lab. The solution GCE provides...

...other RNA components are required? CasPEDIA provides information on crRNA and tracrRNA requirements, along...field has exploded with dozens of Cas enzymes and variants available. Without a comprehensive resource, it... and cleaves ssDNA in trans. In sum, CasPEDIA documents the target substrate (DNA/RNA and strandedness...(nick, bind, cleave, mixed). This information represents the ‘big picture’ features of the enzymes documented...a candidate Cas for your experiment. Target requirements As we all know, a Cas guided by a gRNA is useless...) in some cases. The resource displays these requirements in regard to their sequence (NGG, etc.) as well...experiment. Perhaps you are a structure junky who just wants to learn about Cas domains. CasPEDIA either directly...

...the most recent advances and how some of these variants may be appropriate for your specific research ...nuclease When choosing the Cas9 nuclease for your experiments, there are three important factors to consider...isolated from different organisms and engineered Cas9 variants that were able to target different PAM sites (... be targeted (Hu et al., 2018).  These altered variants can allow for targeting of your genomic loci of...sensitivity in choosing the Cas9 to use in your experiments   The CRISPR drawer. So many options! ...off-site activity of Sniper-Cas9 to different Cas9 variants using gRNAs of different lengths (Lee et al., ...suitable for in vivo research and potentially treatments. Cas12b, characterized by the Zhang (Strecker...

...domain from PE6e-g produced additive improvements. These variants are worth testing if you want to make...Over 75,000 pathogenic genetic variants have been identified in humans and cataloged in the ClinVar database... potential to correct only a minority of those variants in most cell types. But prime editing, a CRISPR...goal of the experiment can all impose different constraints and affect how you select and design your prime...research, the Liu lab created and evaluated 19 PE1 variants with RT mutations known to increase activity, ...system as complex as prime editing, every efficiency counts! The optimized enzyme can be used with any of the...PE2max, PE3max, etc. PE6: Specialized prime editing variants Four years after their initial publication, the...

... of the party goers, the piñata bursts and its contents spill to the ground. There is something akin to... trouble is, with so much choice, in those few moments after the piñata burst, value was subjectively ...disconnect arising between those reviewing the contents of the piñata and the audience blindly receiving...proceed in the future. When we think of designing experiments using CRISPR, there are two major areas where... The foremost handles the design of sgRNA and represents the lion’s share of the currently available tools...example is CRISPR-GA. These tools assess repair events by type (NHEJ/HDR) and track indels at a single...more common. User interaction: Software design elements that fall into this category describe how the ...

...bind to the operator. The bound repressor then prevents transcription from occurring, which means that...), TetR preferentially binds Tet over the TetO elements, allowing transcription to proceed (pJKR-H-tetR... virus. tTA binds to promoters containing TetO elements (often linked in groups of seven as a Tet Response...binds tTA, resulting in a confirmation change that prevents binding to the promoter and turning transcription... Bujard, both the promoter and transactivator components of the system have been engineered and improved...Tet-Off vectors (Tet-Advanced)” are the most recent variants of the Tet-Off system.  For more information on...binds cTA, resulting in a confirmation change that prevents binding to the promoter and turning transcription...

... can start your experiment? What about all the reagents you need to order? Sometimes, it feels like an... Doench also calculated the cost of consumable reagents used to make a plasmid, which came out to $115...means that the same plasmid would cost $155.25 in reagents and consumables today. Finally, this doesn’t account... the scientists designing and performing the experiments. This cost varies hugely based on a number of...assume it’s at least equal to the $155 spent on reagents. Five ways to decrease plasmid production time...plasmids with high-throughput methods that save on reagents, consumables, and time. We also handle the MTA...order to preserve time and money for their own experiments. Keep good records Oftentimes, you’ll be able...

...the risk of such events, newer lentiviral packaging systems divide the viral elements among multiple plasmids...Replication competence can arise from recombination events during viral production and poses a biosafety risk...carrying the transfer gene. By dividing the viral elements across multiple plasmids, the risk of recombination...for transfection can make these optimization experiments a bit more challenging. With these considerations... thereby increasing the number of recombination events that are required to generate RCL. To overcome ...drive high levels of expression of certain viral components; according the manufacturer these changes result...lentivirus, the plasmids encoding the structural elements, envelope, and transfer gene are transfected into...

...measure these properties, and what fascinating experiments could push these tools even further? All of these...and find further tips for using FRET in your experiments here.     What is this FRET you speak of? Förster... acceptor In order for FRET to occur several constraints must be met. The donor and acceptor fluorophores...improved dynamic range for fluorescence lifetime measurements." PloS one 10.8 (2015): e0134436. PubMed PMID...the probe. More on these biosensors later on). Experiments using intermolecular FRET probes are typically...reference standards can be used to validate FRET measurements and serve as a type of positive control. In ...questions about cellular processes. Currently, FRET experiments can probe protein-protein interactions, measure...

... Figure 1: SARS-CoV-2 proteins and other components. Image from Maya Kostman for the IGI.     ...COVID-19 research in other labs. Spike protein, variants, and pseudotyping The SARS-CoV-2 virus depends...cellular receptor. To more easily study the spike variants, scientists have developed ways to pseudotype ...lab. Jesse Bloom’s lab published a protocol and reagents for pseudotyping lentivirus with the SARS-CoV-... has had many opportunities to evolve. Several variants are now circulating and plasmids containing these...pseudovirus and those encoding different spike protein variants that can be incorporated into pseudoviruses. The...at neutralizing the different SARS-CoV-2 spike variants. You can learn more about their research in this...

... but these reagents and strategies are not available to those working with yeast or plants. Using MoClo...the basics of what MoClo is and what types of experiments it enables. What is modular cloning? Modular ...assembly. Simply put, it assembles individual DNA components into larger units or circuits with functional...and a ligase are used to assemble multiple DNA fragments together in a one-tube reaction. Even though most... test a variety of different orientations and components within the system to maximize production potential...multiplexed gRNA systems as well as additional components such as selectable markers, promoters, enhancers...is especially useful for more complex CRISPR experiments (e.g., many guides/parts delivered) in certain...

...organize events that bring different voices together to talk about genetics. In addition to events that we...improved understanding of how our bodies work, new treatments for debilitating diseases, and maybe even ways...individual consumers of technologies and as societal participants in the political process. My role at pgEd I ...readership, and working with middle and high school students. One of these organizations was pgEd. After obtaining...industry conferences, as well as schools, community events, churches or mosques. On occasion, we also get ...researching and writing – following the latest developments in genetics research, their applications and...landscape of genetics and our consistent stream of events mean that there is never a dull day in the office...

...decisions. In my work as a thesis coach for graduate students, I heard about supervisors in all of the above...also considering the needs of other people. Some students and postdoctoral fellows are hesitant about expressing...help you to become more assertive and resolve disagreements with your supervisor, even if he or she is a...will meet both of your needs. 6) Put important agreements in writing One of the most frequent sources ...approximate timelines. Be sure that you keep your commitments, and if for some reason you are not able to, ...they can count on you to follow through on your commitments. Assertiveness is a skill that will help you ...supervisors might try to intimidate you with negative comments (hostile-aggressives, complainers, know-it all...

...technologies have transformed the palette of experiments possible in a wide range of organisms and cell.... elegans approaches rapidly enrich for editing events without the need for any selective marker to remain...the awesome power of genetic selection. Knock-in events tend to be rare, and the challenge with any experiment...in any genetic background, but require variable amounts of experimental manipulation and screening.  A ...screening 768 F1 animals). Recovering rare editing events in a sea of unedited animals struck me as a “needle...into the homologous recombination repair pathway. Reagents required to perform pha-1 co-conversion are available...knock-ins (Ghanta et al., 2020). Together, these improvements from the Mello lab resulted in strikingly high...

...with different fusion proteins for different experiments over the years — a green fluorescent protein,...choose different colors or ligands for different experiments without having to clone a whole new construct...Live-cell labeling strategies. The green structure represents a fluorophore or other useful ligand, gray structure...HaloTag is actually HaloTag7, and recent specialized variants run up to HaloTag11. HaloTag is now the most popular...the results right away. Or perform pulse-chase experiments by sequentially adding different ligands to the...between different colored ligands at different time points.   Figure 3: Single-molecule tracking of...assembly-regulating proteins in vitro reveals complex events. a) Micrographs of a fluorescently-labeled actin...