... Figure 2: Examples of simultaneous multiplex IF labeling of rat brain samples from the James Trimmer...Figure 1: Schematic of multiplex immunofluorescence approaches. A) Sequential Multiplex IF, where one target...you will need to start with a fixed sample. Depending on the sample type, this might be frozen or formalin-fixed...al, 2022 for a sample protocol.)  Simultaneous IF Depending on your needs, your sample, and targets, you..., or you can make your own. For example, to label the rat brain sample shown in Figure 2A above, Andrews... get completely washed away and leave no signal. If you see lots of extra signal in these samples where...set of samples where you label only one target in each. Compare these individually-labeled samples with ...

...for multiplex genome editing.  For a few reasons, Cpf1 is a simplified system for editing multiple targets...protospacer Multiplexing CRISPR-Cas9 options before Cpf1 crRNA array Prior to this new Cpf1 multiplexing method...allow for expression of multiple gRNAs (i.e. Csy4 cleavage sequence, tRNAs, multiple individual promoters...Cpf1 counterparts. Table 2. Cas9 Multiplexing Options  Multiplexing Method Delivery Method Advantages...Xie et al  Multiplexing crRNA expression with Cpf1  The key advantage of multiplexing crRNA expression... chart below for a summary of multiplexing efficiency in vivo. Multiplex gene editing with CRISPR-Cpf1... CRISPR technologies.  It’s a simple and effective method with multiple applications (in vitro, in vivo...

... cloning techniques with higher efficiency and simpler procedures have been developed. Some of these include... excessive time requirements. FastCloning is a simpler yet reliable cloning technique that was developed...insert with an additional 15-17 bases that are complementary to the overlapping vector ends. See an overview...an hour at 37°C. This should be enough time to completely digest the template DNA. Note—templates must ...

...sites. Modifying multiple genes at once. Using multiplexed gRNAs can target multiple locations in the ...CRISPR makes it easy to target multiple loci - a concept called multiplexing. Since CRISPR is such a robust...you add multiple gRNAs on one plasmid. Sound good? Addgene has many tools to help you multiplex - we’ll...Drosophila, and zebrafish!   Why use multiplexed gRNAs? By expressing multiple gRNAs on the same plasmid, you...clone and transfect multiple promoter-gRNA constructs in order to target multiple sites! In this post,...we'll cover Cas9 multiplexing options, but also check out our blog post about multiplexing with Cpf1.   ...  Figure 1: Multiplexing allows researchers to express multiple gRNA from a single construct. DNA...

...end up with poor droplet quality. Using a droplet generator, the sample and the droplet generation oil ...take place.  When the PCR is complete, a droplet reader extracts the droplets from the plate and measures...reagents (and time)! AAV titering using droplet digital PCR Droplet digital PCR involves partitioning a PCR... approximately 20,000 droplets using water-oil emulsion technology. Each droplet contains the ingredients...emulsion containing approximately 20,000 droplets. Each droplet contains the material required for a mini...each one. Droplets that fluoresce contain the amplified target sequence. After all of the droplets have been...negative (gray) droplets. The no template control wells should have very few positive droplets. In the image...

...scalability and efficiency of multiplex gene editing. Moreover, existing multiplex strategies have been facing...developed DAP arrays for efficient multiplex base editing (MBE) and multiplex prime editing (MPE) with only...Schematic of multiplex gene editing with DAP array   Use DAP arrays can enable efficient multiplex gene ...array for multiplex gene editing applications, we provide detailed manual and online design examples of using...natural CRISPR locus of a bacteria host encodes multiple guide RNAs (gRNAs) on a single array to target...leveraged such host-defense mechanisms to enable multiplex gene editing in a variety of cells and organisms...from a single array, has been a top choice for multiplex gene knockout or regulations, it lacks proper ...

...Fluorescent proteins (FPs) are also the foundation of multiple important technologies, including FRET and optogenetics...treatment can photoconvert GFP from green to RED! This simple technique has been shown to work in plant, Drosophila... like PsMOrange and mEos add another layer of complexity to imaging, as they allow one to monitor compartment...

...dysregulation event. Diseases are often complex, with multiple layers of dysregulation from either genetic...to each cell, future applications could deliver multiple TFs to each cell, allowing for combinatorial overexpression...the specific dysregulation in the disease. For example, very few diseases manifest as a single mutation...

...author “Coping with Difficult People,” identified seven types of difficult people: Hostile-aggressives, who... are a few principles that will help you resolve most conflicts, even with difficult people. If I had ...supervisor, or do you have to work with difficult people? Each time I ask this question at my workshops,...had to use one word for to summarize these principles it would be “assertiveness.” 7 strategies to become...confidently while also considering the needs of other people. Some students and postdoctoral fellows are hesitant..., especially if their supervisors are difficult people. The following seven strategies will help you to...frequent sources of conflict is miscommunication. For example, you might misunderstand your supervisor’s suggestions...

... been used to date . If you have any questions, please write to us in the comments section.  FX cloning...important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803...directly engages the ribosome for translational complex assembly. PhD student Dejan Gagoski used SITS ... research. If you’d ever like to deposit a kit, please contact us at [email protected]. ...

...design GGA overhang sets, particularly for high-complexity reactions? The researchers over at New England...data-driven approach might allow for an increase in the complexity. In 2018, they published a paper profiling four...2022).     Figure 1: Workflow for high complexity Golden Gate Assemblies. Image courtesy of New... three DAD tools used were key for ultra-high-complexity assembly. While the traditional rules of thumb...data-driven approach was needed to massively expand complexity. The three tools developed, outlined below, allow...traditional design rules) for successful high-complexity Golden Gate Assembly reactions. NEBridge Ligase...of overhangs, stat? GetSet can do that for you, complete with predicted fidelity. You can specify number...

...will be necessary to improve its multiplexing capacity. Multiplexing is key due to the natural redundancy...graphic below - including some simple comparisons. The easy multiplexing afforded by PTGs will improve...knockout, or multiple gRNAs may be used to target genes with overlapping activity. Multiple gRNAs can also... Addgene Other Techniques for gRNA Multiplexing Multiplexed and programmable regulation of gene networks...;  to observe a phenotype, the modification of multiple genes is often necessary. Guide RNAs (gRNAs) are...editing efficiency. One previous strategy for multiplexing gRNAs used the RNA nuclease Csy4 from Pseudomonas... test the application of this system to gRNA multiplexing, Xie et al. constructed PTGs containing 8 gRNAs...

...see their name tag if you are one of those people. Meet people in sessions by sitting next to someone and...conferences. Conferences provide the opportunity to meet people who are interested in the same things you are on...will meet a potential collaborator Some of these people you meet will review your papers or grants in the...the future, and don’t be surprised to know that people think more favorably of scientists they know personally...depend on your national recognition and the more people you know, the larger your profile in the community...opportunities but can be overwhelming as far as meeting new people. At large conferences, look for smaller events ...media to let others know you will attend. Meet some people online to follow up with in person. Email lab members...

...IBEX is a highly multiplexed tissue imaging method that uses iterative staining and chemical bleaching...Radtke et al., 2020, 2022). I do mean highly multiplexed: you can image over 65 different parameters using...community and the database. They particularly encourage people to upload and share their negative data. After ...an invaluable resource for anyone performing multiplexed imaging, not just IBEX. The IBEX Imaging Community...., & Germain, R. N. (2020). IBEX: A versatile multiplex optical imaging approach for deep phenotyping ...phenotyping and spatial analysis of cells in complex tissues. Proceedings of the National Academy of Sciences, 117...resources on the Addgene blog Antibodies 101: Multiplex Immunofluorescence Negative Can Be Positive: Open...

... in proximity by the looping of chromatin, the complex of DNA and proteins that forms chromosomes. These... of the CAPTURE method. After protein and DNA complexes are cross-linked, DNA is digested into smaller...and also makes it hard to compare results across multiple targets because different CAPTURE cell lines will...and 2) gRNAs Number of loci targeted One Many Sample type(s) Cell lines Cell lines, primary cells ...lab presented three ways to use CAPTURE 2.0: 1. Multiplex capture of chromatin interactions The Xu lab performed...experiment to show that CAPTURE 2.0 could detect multiple chromatin interactions at once. Chromatin interactions...Long-range chromatin interactions identified with the multiplexed CAPTURE 2.0 largely replicated interactions previously...

...replicate.   How can I prevent recombination in my samples? Plasmid recombination can affect viral vectors...vectors (LTRs and ITRs), plasmids with multiple polyA sites, and other plasmids that contain repeating elements...stable E. coli reduce the rate of recombination in samples. For a more detailed comparison of these cloning...following protocols for low copy plasmids. For example, using a longer growth period (up to 24 hr), reducing...rich media can improve growth and DNA yield. Test multiple, small colonies After streaking LB agar plates...cultures contain the recombined vector backbone. Test multiple colonies before saving a glycerol stock to ensure... easiest and clearest ways to determine if your sample has recombined. Choose a restriction enzyme that...

...ensure that samples are easy to locate in freezers that may be shared with multiple people or even multiple.... Some samples are submitted as bacterial colonies in petri dishes, but close to 80% of samples are received...of the samples that come through our doors. It would be a nightmare if we didn’t have a simple way to ...every sample can be tracked easily at each step in the storage process. After the original sample is barcoded...scanned to verify that it is the correct sample. Tracking samples - it's all about location, location, location...these samples have the 2D barcodes mentioned above. These barcodes identify what type of sample is contained...allow us to store multiple types of tube for each plasmid we have at Addgene. Each sample at Addgene has ...

...affected the pH of our viral samples. Read on to learn how aqueous samples might be affected by dry ice...Measuring the pH of our samples: What we did To measure the pH of our samples, we used a solution of cell...when the samples were frozen. This was clear by the gradual change in pH visible at the sample interface...this, we stored samples on dry ice for 72 hours and then vented the tubes by completely removing the caps...at -80°C. For samples that are shipped on dry ice, this can affect the pH of the sample, which may have...have some effect on the contents of the sample. For example, one study found that dry ice storage affected...describing how short term storage on dry ice can shift sample pH, we wondered whether or not the dry ice we use...

... one simple experiment. PCA is an in vivo technique where two proteins are fused to complementary reporter...neighboring proteins. Thus to capture protein complexes, scientists can simply express a bait protein ...suboptimal folding or stability. Thus they conducted multiple rounds of directed evolution until they identified...interactions Split-BioID combines a protein fragment complementation (PCA) technique with proximity labelling to...labeling and purifying additional proteins from a complex of interest. For more information, read the Split-BioID...detect RNA-protein interactions that can regulate multiple biological functions. RapID (RNA-protein interaction... and Paul A. Khavari. "Research Techniques Made Simple: Emerging Methods to Elucidate Protein Interactions...

... have no fear - I’m here to introduce you to a simpler, yet more effective, CRISPR-based epigenetic approach...machinery.   Well, I’ve got some good news: complete DNA demethylation can be achieved by dCas9 alone...Sapozhnikov and Szyf 2021).    Figure 3: A simpler approach - dCas9 blocks DNMT1 methylation, leaving...too.  High dCas9 and gRNA expression are key for complete DNA demethylation – tight binding is crucial for...blasticidin or puromycin resistance genes.  It’s that simple! Good luck and happy editing!    Thank you to...