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...comparison of this one-step staining with nanobodies vs two-step staining with antibodies. Nanobodies...before application to the sample. Staining with this multi-color staining workflow yields similar localization... fusion with a reporter enzyme. For multicolor staining, careful consideration must be used when selecting...different IgG subclasses of 1° for multi-color staining Can be expensive Nanobodies   Recombinant...together. At the bench, this results in a shorter staining protocol because the primary antibody can be “...Nanobodies also simplify multicolor staining experiments because they don’t require using primary antibodies from...below, triple colocalization is possible even when staining cells with three IgG1 antibodies. Antibodies were...

...number of challenges ranging from long periods of training to limited job opportunities in academia - the...the main reason most people enter postdoctoral training. Similarly, there are several factors to consider...about how to get the most out of your postdoctoral training experience. If you're going to commit to doing...doing a postdoctoral fellowship, what are you signing up for and how can you do it well? Below, I briefly...and create a timeline for achieving these goals. Planning is often more easily done in reverse. Assuming...before the position would begin- for you, at the beginning of Year 5. Now work backwards. To publish a submitted... critical to get an experimental system up and running very early on. Once your system is working, you...

...gram negative bacteria, staining them red or pink. At the end of the staining procedure, gram positive...Streptococcus pneumoniae, while the alcohol used in the staining process bleached another, Klebsiella pneumoniae...expanded on Gram’s discovery, adding a second stain, safranin, as a counterstain to help visualize the bleached...and immediately rinse. Flood the smear with the safranin counterstain for 30 - 60 seconds. Rinse the slide...positive (purple) and Gram negative (red/pink) staining. Image credit: Mark Perkins https://www.flickr.com...check out these resources.   Bruckner, M. Gram Staining. https://serc.carleton.edu/microbelife/research_methods...creating a complex within the peptidoglycan layer, staining the cell purple. When the alcohol is added, it...

...are ready to begin the screening process. Want to set yourself up for a screening success? Before you start...’s best to do some planning in the design phase of your experiment for a screening strategy. Restriction...looking for your edit of interest. For all of the screening strategies outlined below, the technique can be... isn’t a multiple of three will do. A popular screening method for knockouts is Tracking of Indels by ...frequency of your sgRNA-Cas9 combo. Pro tip: When designing your PCR, ensure there is at least ~200 base pairs...deletion large enough that it can be visualized by running your PCR on a gel. It’s still paramount that you...using a dual guide system.   Fig. 2: Size screening for large deletion events.   Validating knock-in...

...functional analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key... the plasmid containing the mutation). Similar results can be obtained with PCR screening using the primers...manner. The approach can be used in conventional cloning (to introduce or remove restriction sites), in ...final circular plasmid product pictured here. B) Screening of recovered plasmids for the mutation of interest... vastly facilitates the subsequent process of screening for succesfully mutated clones. Forward and reverse...Taq generate A-overhangs (a feature used in TA-cloning). Such non-complementary bases at the 3’-end interfere...can also form colonies under these conditions. Screening If a restriction site was introduced (or ablated...

...of whole phages as therapeutics, which is also gaining a lot of new traction lately (1, 2), depends on...varied by the cell and whose structures are just beginning to be understood. Therefore, in addition to their...identifying and characterizing sugars and sugar-containing molecules in molecular biology labs (for a review...binding proteins are hard to find Sequencing and cloning phage DNA can be difficult, in part because phages...Nebulization involves forcing DNA through a small opening using compressed nitrogen gas; this shears DNA ...RBP gene without causing issues associated with cloning large fragments. After fragmentation, repair sheared... colonies using primers bordering the multiple cloning site of the vector. In order to reduce the chances...

...especially with my deep learning-wizard colleague Ahmed Allam. Continued training led to the recent development...High-throughput screening of pegRNA efficiency and building prediction tools with machine learning (PRIDICT,... scaffold sequence, and an extension sequence containing a primer binding site and a reverse transcription...al., 2024). The additional datasets used in the training of PRIDICT2.0 make this model more suited for ...your target. Figure 3: High-throughput screening of the effect of chromatin on prime editing efficiency...prediction tool (ePRIDICT). In summary, machine learning-based prime editing prediction models (such as...biology. His expertise includes applying machine learning techniques to make sense of biology, with a special...

... an education . . . is not the learning of many facts but the training of the mind to think something ...take a different approach. His own most impactful learning experiences happened in the lab, and he wanted... work and become more actively involved in the learning process. As trainees become more comfortable with...interest in scientific career paths while also broadening access to experiences that promote professional...trading structure and predictability for improved learning outcomes and the opportunity to make new discoveries...teaching style, with the former offering excellent training in improvisation and the latter making him a more...by measuring NADH production.   CUREs are beginning to transform undergraduate education in the sciences...

.... P., White, M. F., Yakunin, A. F., Garrett, R. A., Van Der Oost, J., . . . Koonin, E. V. (2015b). An ... M. P., Venclovas, Č., White, M. F., Yakunin, A. F., . . . Koonin, E. V. (2019). Evolutionary classification...classification system for the array of CRISPR technologies. Learning the general principles of this classification ...S., Oshiro, R., Makarova, K. S., Macrae, R. K., Koonin, E. V., & Zhang, F. (2023). Uncovering the functional...Chylinski, K., Makarova, K. S., Charpentier, E., & Koonin, E. V. (2014). Classification and evolution of ...1314. https://doi.org/10.1134/s0006297921100114 Koonin, E. V., & Makarova, K. S. (2019). Origins and evolution...20180087. https://doi.org/10.1098/rstb.2018.0087 Koonin, E. V., Makarova, K. S., & Zhang, F. (2017). Diversity...

... bystander edits—unintended changes to nearby target bases. Up to 5 bystander adenine bases were edited...Base editing was invented in David Liu’s lab by combining Cas9 DNA-targeting technology with a fused cytidine...reduced RNA editing to background levels while retaining acceptable on-target DNA editing activity. In ...was an important addition. Improved activity of adenine base editors The first ABE was about 50% efficient...and longer genome search times. Using a machine learning approach, the lab proposed multiple sets of Cas9...all bystander edits produced synonymous changes, meaning the new codon corresponded to the same amino acid...& Ciaramella, G. (2020). Directed evolution of adenine base editors with increased activity and therapeutic...

...for Host Binding Proteins & Running for Rare Disease, Running for FOP, Running for AJ I have two picks for...the post. The second post, “Running For Rare Disease, Running for FOP, Running for AJ,” was contributed ... we’re raising money for a cause while running. I love running and this event gives me the opportunity...sitting in the San Francisco International Airport listening to the Lion King soundtrack and writing this post...Project That’s it for now. As I indicated in the opening paragraph, I’m currently on my way back from a ...

...generate gRNAs and homology arm repair templates containing the tag for each insertion. Also, it remains ...simultaneously introduce a selection marker for screening modified strains. With SapTrap, the user first... (Fig. 1, Step 1). There is no need for PCR or cloning, as digestion of the destination vector with SapI...SapTrap includes a prebuilt donor plasmid library containing several types of fluorescent (EGFP, tagRFP, mCherry...selectable marker (floxed Cbr-unc-119) for easy screening of the insertion event, and a variety of connector...hundreds of genes, generating powerful genetic screening libraries. Additional protein tagging systems ...reading frame of the insertion, and 3) a vector containing the desired tag, which can be obtained as a universal...

...microhomology-mediated end-joining)? MMEJ, a form of alternative end-joining, requires only very small ...research, you know all about using non-homologous end-joining (NHEJ) to make deletions or homology-directed repair...month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction There...repair template is present. HDR also presents a cloning dilemma - the longer the homology, the more efficient...with longer homology arms comes more time spent cloning. In contrast, the short homology required by MMEJ... regions of homology, which facilitates easier cloning. An overview of PITCh. The PITCh plasmid...inserted into the PITCh vector via In-Fusion or SLIC cloning. Step 2: Design an insertion locus-specific gRNA...

...defective allele Ym and you need to convert an adenine (A) to a guanine (G)? You may wonder if a CRISPR-base editor...molecular components. Adenine base editors for plant We developed plant adenine base editor (ABE) vectors...with the desired outcome are not pictured. Adenine base editors (ABE) What if you have another gene...deaminases' which would theoretically give rise to an adenine base editor. A massive protein engineering effort...an adenosine deaminase that can act on DNA for adenine base editing. They first created a defective chloramphenicol... (H193Y). The reversion of this mutation by an adenine base editor would restore antibiotic resistance...into E. coli harboring the defective CamR gene. Screening for surviving colonies and subsequent rounds of...

... in plasmid cloning Perhaps the most widely used application of CcdB is GatewayTM cloning, where the ccdB...toxin is stable while the antitoxin is unstable, meaning that the cells must continually produce antitoxin... not inherit plasmid due to improper plasmid partitioning, it will still inherit some toxin and antitoxin...counterselectable marker in the destination vector. Successful cloning occurs when the ccdB gene is replaced by the gene...modifications in the yeast Pichia pastoris. By combining both positive and counter selection in one plasmid...plasmid, they were able to create an efficient cloning tool. The plasmid pKSCTMF contains the mazF gene...retained in less than 10% of cells while plasmids containing the plasmid addiction system were retained in...

...to select against CRISPR positive plants, but screening these can be challenging and requires specialized...requiring the destruction of plants or tissue for GUS staining or are not suited to studying large samples. RUBY-Red...sites. These convert the amino acid tyrosine to betanin, a member of a class of pigments primarily found...order Caryophyllales, such as cacti and beets. Betanin belongs to a different chemical family than the...purple plant pigments found in all other plants, anthocyanins. Figure 1: An adult plant expressing...selecting transformation events and am currently planning to use it to simplify my work with an Arabidopsis... to study these mutants.   Fig. 2: (A) Betacyanin is produced from tyrosine by the activity of ...

...are crisp, bright, and lacking in non-specific staining can be a challenge in the best of cases. But it... antibody, resulting in significant background staining (Figure 1A). Not all tissues will have the same...researchers can get away with species on species staining with no problem! But to be sure that your secondary...experiments. If you do find high levels of background staining by the secondary, then the following three strategies...help!     Figure 1: Species on species staining can be a problem because the secondary antibody...that you can significantly decrease background staining by mixing your primary and secondary antibodies...are a few common solutions to avoid background staining when doing species on species IHC. (A) One solution...

... would not hide behind these two fingers the ninety-nine cubits of Aristotle, nor would you mention my...where our medicines, technologies, defense, and governing policies are developed primarily by wishful thinking...culminated in the current President holding and campaigning on many views that don't line up with the scientific... us. I worried the march would be a simple, unquestioning celebration of science, at a time when scientists...Jennifer Stancill I am a research biologist by training, so of course I felt compelled to participate ...say that I was supremely proud of Nashville that morning. Despite the rain, about 4,000 supporters of science...one person vocally protesting us, he was not threatening in any way. Participating in this march proved...

...postdoctoral pay/benefits commensurate with work training and experience, better align trainee expectations...temporary and defined period of mentored advanced training to enhance the professional skills and research...Associations around Boston - From Panel Session 2: Defining the Postdoc: An Institutional Perspective. This...of the academic research workforce, and we are beginning to identify potential solutions. Keynote speaker...structural problems seem to boil down to that fact that training in the academic research environment is setup ... is this very idea that postdocs are acquiring training to become professors that is provided as an explanation... they don’t make in wages, they make up for in training. Unfortunately, these low wages also make it so...

...imaging of cellular targets Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...frankenbody (FB) construction from the complementarity determining region of an anti-FLAG scFv and a stable scFv...small epitope tag (FLAG is eight amino acids, HA is nine), multiple repeats of the tag can recruit multiple...10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...Gabrielle Clouse While many CRISPR-based genetic screening methods have primarily used Cas9 to knock out ...the Broad Institute have expanded the genetic screening toolkit by optimizing a Cas12a-based method for.... 2021 To validate this optimized genetic screening technique, researchers identified a synthetic ...