...Figure 1: Opto-katanin induces fast, localized microtubule severing. A) Schematic of Opto-katanin constructs...microtubule-severing protein, Katanin is of course named for the samurai sword called a katana. Opto-katanin consists of...use. And now, onto our optogenetic tools!  Opto-katanin: a light-powered microtubule-cutting sword Microtubules...tool for localized microtubule disassembly, Opto-katanin (Meiring et al., 2022), that overcomes many of ...EB3N-VVDfast-iLID (anchor) and SspB-mCherry-p60 (katanin) are recruited to microtubules under blue light...from the microtubule body. B) Schematic of Opto-katanin’s use in cells. Images adapted from Meiring et al...microtubules and iLID-SspB to control the recruitment of katanin to that anchor. So, you’ll need to express both...

... thought about running one, you’ll know there’s a lot of choices to make when designing the experiment...for SDS-PAGE running conditions and will cover most western blot use cases. If you’re running native (non-reduced...of gels  Gel Type Protein sizes Running buffer Running conditions Pros Cons Tris-glycine...Figure 1: The technical decisions to make when designing a western blot.  Psst! Looking for a western...lyse and denature them. If you are intentionally running non-denatured proteins, in what is known as a native...proteins. For this post, we are assuming that you are running a denaturing blot.  There are a number of lysing...cytoplasm or whole-cell lysate. A harsher, SDS-containing lysis buffer, like RIPA, should be considered...

...genomic fragment containing the sgRNA target sequence can be placed in multi-cloning site (MCS) between...floating around, and many pre-designed plasmids containing these variants ready for purchase. But what is...post will help get your in vivo research up and running as soon as possible! Selecting your target CRISPR...has been quite successfully adapted for use in screening experiments as well). Therefore, if you’ve started...to attack this gene. Should you cut it at the beginning of the coding sequence to delete the entire gene...previously validated gRNAs at Addgene. In vitro screening for the best gRNA Figure 1: Validation ...plasmid was co-transfected with pX330 plasmids containing sgRNA sequences into HEK293T cells. The fluorescence...

...including vector-insert joining for recombinant plasmid construction (restriction cloning), adaptor ligation...Learn New Plasmid Cloning Techniques Pushing the Limits of DNA Assembly Plasmid Cloning by PCR Resources...ligases are enzymes that seal breaks in DNA by joining 5 ́-phosphorylated DNA termini to 3 ́-OH DNA termini... is highly active in nick ligation and DNA end-joining, but will also efficiently ligate many undesirable...undesirable structures, including substrates containing gaps or DNA base pair mismatches (8-11). For some applications...applications, there cannot be any end-joining activity, and for others, there is a need for the exclusive...DNA Ligase, which only reacts with substrates containing no gaps, and will not join any fragments end-...

...secondary antibody) approach?  Staining for multiple targets When staining multiple targets indirectly, ...for a four-antibody panel, staining in parallel    In short, when staining multiple targets, look for...will recognize and bind to your primary antibody. Manning et al. tested a variety of commercially available...there was a detection bias of IgG2a>IgG2b>IgG1 (Manning 2012).      Figure 1: Broadly reactive...depends on the application and target. If you are running a basic western blot and probing a single target...mouse tissues, causes a high degree of background staining. This occurs because the anti-mouse secondary ...the tissue sample. There are species-on-species staining protocols that use antibody fragments to block...

...More complex 24-fragment assembly Single insert cloning efficiencies were measured based on the acquisition...Antibiotic selection allowed for high throughput cloning efficiency measurements. We performed additional...suppression of background by antibiotic selection. Screening of transformants by colony PCR confirmed the insertion...presence of every module, destination construct and functioning Type IIS restriction enzyme and DNA ligase, any...obtained if any single component was omitted. Screening for successful Golden Gate Assembly All Golden...researchers can plate larger volumes of the outgrowth containing the transformants onto the selection plate. Figure... Ligase, the mainstay of most biotechnological cloning efforts for over 50 years, prefers ligation of ...

...that many researchers feel that they lack formal training in a variety of transferable skills. At Addgene...huge asset! As is true for any skill, there is a learning curve for collaborating and it is always a good...perfect platform for practicing initiative and for training in teamwork and collaboration management. For ... email and social media (see the section on “Maintaining Relationships” below). Expand: Collaboration ...complex projects. It is also fun! I really enjoy learning from others and meeting people with different ...was very difficult and somewhat awkward in the beginning. But, as so often happens, one gets better with...people outside the university within the context of learning and, in my experience, mistakes and insecurities...

...populations, as well as a pragmatic approach to determining the selectivity of antibodies for other sample...found that some antibodies perform well in a PFA-saponin fixation and permeabilization protocol, but very...usually test 3 different fix/perms (methanol, PFA-saponin, PFA-triton). The fix/perm solutions can make a... your antibody. When compared, 4% PFA and 0.1% saponin; 4% PFA and 0.1% Triton X-100; and methanol, we... and fixed in 4% PFA and permeabilized in 0.1% saponin. 400,000 cells were stained with the indicated ...CoraLite® Plus 647 secondary antibodies. Antibody staining was quantified using the Attune NxT Flow Cytometer...Cytometer with representative images showing the staining intensity in the KO population (pink histogram)...

...causing it, try making fresh running buffer. Old or improperly made running buffers can cause all kinds...transfer worked with Ponceau S staining or other reversible protein staining immediately after your transfer...troubleshoot a western, you’ll need to be familiar with running a gradient, which will allow you to optimize the...can help answer your particular question before running one.  Good on gradients? Excellent! Now, let’s ...subcellular compartment, you may want to try an SDS-containing detergent like RIPA. You can try a few different...V/cm of gel), your gel may be overheating. Try running it in a cold room or putting ice packs in and/or...kinds of odd issues. If your gel is running properly but you’re not seeing sufficient resolution in the bands...

... them not working, or spend a few weeks designing and cloning the vectors you actually want. But now you...Plasmids 101: Modular Cloning Addgene's CRISPR Guide Plasmids 101: Golden Gate Cloning...designed for a quick, plug-and-play approach to cloning CRISPR vectors (McGee et al., 2024). It reduces...truly is plug-and-play. Because of its modular cloning approach, it’s easy to switch out reporters, Cas... overview and timeline of the Golden Gate (GG) cloning approach. Individual modules and a sample ligation...them into a destination vector via Golden Gate cloning, perform an exonuclease cleanup, transform, plate...experiment. As always, we recommend checking your cloning via a miniprep and restriction digest, and then...

...PCR amplified for cloning into one of three Zeamays codon-optimized Cas9-containing binary vectors. The...introduce you to some of your potential options and cloning methods, but please scroll down for plasmids suitable... can be inserted using Gibson Assembly or SLIC cloning methods. A BsaI-based E. coli multiplexing plasmid...together and drive ordered assembly into a Cas9-containing destination vector. Note: depending on which ...four unique kanamycin-resistant plasmids, each containing a different gRNA target sequence downstream of...digested using BsmBI and ligated into Cas9 or dCas9-containing destination vectors. Destination vector options...), and dCas9-VP64 (transcriptional activator)-containing plasmids. Each destination vector contains GFP...

...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...