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...process of cloning, combining, and testing these different parts can be quite arduous. The cloning process...of a good screening experiment can be found on our blog post entitled “Genome-wide Screening Using CRISPR...Moduclar Cloning (CIDAR MoClo) Parts Kit. This Kit consists of a variety of plasmids containing many different...compatible with both standard Gateway cloning and the FNLCR Combinatorial Cloning Platform (CCP); the latter of...terminators for easy cloning of genes and promoters. In addition to USER cloning, this toolkit is also...GPCRs in the human genome are orphan receptors meaning their ligands are unknown. Identifying new ligands...approach termed Parallel Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO...

....  The partitioning problem Plasmid incompatibility also occurs because of plasmid partitioning (Schumacher...within one cell have the same replicon and/or partitioning system. Let’s start with the replicon- the part...to initiation replication. However, in iteron-containing plasmids,  RepA can also bind to its own promoter...a concern with low copy plasmids, though the partitioning problem does occur in high copy plasmids (Diaz...are several different versions of the plasmid partitioning system in the bacteria, but generally it consists...For high copy plasmids, incompatibility due to partitioning is similar to incompatibility due to having ...loss (Diaz et al., 2015).  Figure 2: The partitioning problem. For low copy plasmids, the main ...

...genome at once! Knockout screening with CRISPR libraries In CRISPR knockout screening, sgRNAs guide Cas9 to...libraries called Julianna, for mouse screening, and Jacquere, for human screening. These new libraries have improved...inhibition screening with CRISPR libraries Libraries for activation or inhibition CRISPR screening are similar...Schematic of phage display screening with a pooled library. Surface display screening can also be conducted...the single-cell level. CRISPR libraries CRISPR screening has become a popular tool for probing cellular...cellular pathways and mechanisms. CRISPR screening libraries supply a collection of sgRNAs, but they may or ... Figure 2: Schematic of CRISPR knockout screening with a pooled library. Created with BioRender....

...Molecular cloning requires some method of screening colonies for the presence of an insert. Traditionally...then you might consider designing orientation-specific primers. Blunt end cloning is an example of when ... Addgene Blog Learn to do Restriction Cloning Try SLIC cloning Learn about Gibson Assembly Additional...things to consider when performing colony PCR.   Designing colony PCR primers The first and perhaps most ...most important step to colony PCR is designing primers. There are 3 strategies for primer design: 1) insert-specific...backbone. This type of primer pair is also great for screening clones created with the same backbone but that... When you design primers to anneal outside the cloning site, it doesn’t matter what the sequence of the...

...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...

...transitions are edits between the purine bases (adenine, guanine, and hypoxanthine) or between the pyrimidine...glycosylase (MPG), also known as alkyladenine DNA glycosylase (AAG). When adenine base editors (ABEs) were originally...BioRender.com. Inosine excision leads to the first adenine transversion editors Uracil excision is the most...endogenous enzyme is not efficient enough to hinder adenine base editing. Once base transversion editors were... able to edit A → Y (Y = T or C) in 67.17% of screening cells (Tong, Wang, et al., 2023). Based on this... team used rational mutagenesis to produce the adenine transversion editor AYBEv3. This tool still retained...A → C product purity of 48%. Like CGBEs, these adenine transversion editors produce a lot of indels (up...

... 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...

... 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... 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...