...for an example image). Point Scanning Confocal Microscopy Point Scanning Confocal Microscopy was the first...microscopy technique to incorporate optical sectioning. Optical sectioning refers to the ability to extract light... Confocal Microscopy (Spinning Disk) Parallelized Confocal Microscopy (Spinning Disk) increases the speed...to enhance performance. In spinning disk microscopy, a metal disk containing a number of holes rotates ...samples always require some form of optical sectioning. Point scanning confocal microscopes (see above) often...volumes to enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy...excitation light to a point in the sample and raster scanning the point to build up a final image pixel by pixel...

... analytic scientists seem to toss data-driven reasoning out the window when making decisions. We think...decisions without much logic - even choosing a training lab where we will spend the next 6 years. Know...very communal. That didn’t suit my collaborative learning style at all. Finally I rotated in a small lab...about this lab’s reputation? If you are choosing a training lab, I strongly suggest you complete a work rotation... the same field taking projects with them and remaining close colleagues of the PI? Or do lab alumni leave...they “needed it more”. My friend didn’t heed this warning and profoundly regretted it when the same thing...year later. I hear too many science trainees get warnings and still think “but that won’t happen to me, ...

...of scientist training in the US and around the world. There are rumors that we are training too many scientists...opportunities after training. One of the best recent discussions of this apparently worsening crisis recently...the way we view the graduate school and postdoc training years. There is no doubt that there are too many...academia. Certainly funding for academic research and training is getting tighter and competition is fierce. ...sufficiently prepared during their 6-12+ (!) years of training to get jobs. They are not exposed to the vast ...radical (and some not so radical) suggestions for defining the problem and for finding solutions. Even better...meeting new people by working together on the event planning and execution. I am seeing many similar examples...

...reporter. The only caveat is that HA staining is weaker than reporter staining. Picking the right reporter for... them. HRP RANbody staining results in a ≥10-fold increase in signal over staining with a primary/ labeled... sensitive than HRP-RANbody staining Requires secondary antibody staining HA Tagged GFP, RFP, H2A2B ... IgY antibody Staining cultured cells and tissue sections Great for multicolor staining of tissue since... sensitive than HRP-RANbody staining Requires secondary antibody staining GFP, RFP, H2A2B   Are you...smaller size of RANbodies compared to antibodies. Staining with multiple HRP-RANbodies is indistinguishable... domains are especially useful for multi-color staining of tissue because they have limited cross-reactivity...

...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...

...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...

...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...

... 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...

...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 Not all gRNA’s are effective...is to screen you gRNA in vitro first. In vitro screening is a simple and fast method for validating your...gRNAs to Target Mouse Genes To begin the in vitro screening process, you should first develop primers to the...

...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. Abbreviated protocol for PITCh Step 1: Generate...inserted into the PITCh vector via In-Fusion or SLIC cloning. Step 2: Design an insertion locus-specific gRNA...

...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 ...