...components expressed from different plasmid backbones. They noted that backbone differences such as the order,...assess how different lentiviral plasmid backbone elements impact viral titer and to compare different Cas enzymes...Hurt for his expertise and input.  References and resources References Weber, Ernst, et al. “A Modular ...product in an affordable way, researchers test many different combinations of promoters, terminators, solubility...experimental goals are more general in nature, or you aren’t planning on using K. phaffii, then the MoClo YTK...1: A chart with MoClo kit recommendations for different modal organisms and applications.    Plant...be used to test a variety of Cas enzymes with different PAM requirements and editing abilities, such as...

...experiment uses 2 or 3 different fluorescent colors each measuring a different target. Flow cytometers...analysis. Since different populations of immune cells express unique Clusters of Differentiation cell surface...flow cytometry experiments.       References and resources References: Bonner WA, Hulett HR, Sweet RG, ...each cell as it passes through and scatters in different directions. The degree of scatter is captured ...fluorescent signal that is captured by detectors. The strength of the emitted photons varies from cell to cell...highly versatile and some can accommodate up to 30 different colors. This lends flow cytometry particularly...to complex experiments looking at a variety of different targets. If you are planning on using fluorescent...

...Created with biorender.com. Pro tip! If your samples and control underwent different treatment processes...Created with biorender.com. The plot above is for a smeary expression and is quite different from the ...presence or absence, the plots can look quite different. You might have high expression, low expression...shows the population of interest. Created with biorender.com.   In such cases, instead of trying to quantify...yes/no flow cytometry readout. Created with biorender.com.   Using histogram plots to quantify smeary... the mean fluorescence intensity (MFI) of the different populations. MFI is the average brightness from...plot. However, the histogram plots look a bit different than they do for yes-no readouts.      ...

...101 eBook, 1st edition.  References and resources References Swift S, Lorens J, Achacoso P, Nolan GP. ...pseudotype γ-retrovirus using different envelopes to modify tropism. Briefly, different envelope plasmids can ...packaging; however, they are different viruses and thus use slightly different isoforms of these packaging...from lentiviruses? There are a few important differences between engineered γ-retroviruses and lentiviruses...lentiviruses. First, these viruses were derived from different genomes (MoMLV and MSCV for γ-retrovirus; HIV ...be useful for the transduction of terminally differentiated (non-dividing) cells. Both lentiviruses and... in gene therapy research to reduce promoter interference and/or drive high expression of a transgene ...

...nucleus, a different fix/perm kit is required (to be precise, the fixing solution is different while the...decrease in signal strength in a flow plot. d = days post treatment. Created using biorender.com. Cell viability...lineage markers, like many of the cluster of differentiation (CD) proteins found on immune cells. However...antibody host species interactions. Created using biorender.com. Another way of indirect labeling is through...primary antibody host species, as it has to be different from the other antibodies in your panel. Otherwise... dendritic cell, CD8+ T cell. Created using biorender.com.   Intracellular labeling Oftentimes, a ...first fixed and then permeabilized, using two different buffer solutions. Fixing causes protein cross-...

...graph of photocurrent vs. time. The resulting histograms is described through maximum current (H), the...cytometer to increase the electric current and thereby the signal strength. So, we need to adjust the voltage...Gates are the parameters the machine uses to differentiate between variations in those factors. To understand...a little bit of theoretical background on the different parameters that your gating strategy will be based...measured: height (H), area (A), and width (W). The different measures are represented as FSC-A/SSC-A, FSC-H...(see Figure 2). H describes the maximum signal strength, while W results from the time that the cell spent...cells, blood cells and more), each coming with a different size and structural complexity. After several ...

...sequence critical for Cas9 cleavage), thereby rendering the resulting construct resistant to Cas9 induced...that amplifies the entire plasmid template. The parent template is removed using a methylation-dependent...A*” (in addition to the “A” site already in the parent vector). Also shown are the “B” restriction sites...with enzyme “A” as compared to the products of parental plasmid digestion (blue). Digestion with enzyme...mutagenesis. In addition, you may want to try different concentrations of template (e.g. 0.1-1.0 ng/μl...methylation-dependent enzyme (DpnI) to eliminate the parent plasmid from the PCR products, the plasmid template...patch up the DNA. The resistance marker from the parental plasmid provides a mean for selecting for transformants...

...conjugated to different fluorophores or a panel of primaries that was raised in different species or are...are complex and antibodies may perform differently in different samples and experimental conditions. Be...for IHC, not all IHC experiments are the same. Differences in sample processing, fixation, antigen-retrieval...make sure that you can easily distinguish each different type of antibody and therefore each target protein...unique isotype or species, then you can choose different fluorophore-conjugated secondary antibodies specific...product. Note that many antibodies are sold under different names through multiple vendors, so look at epitope...to choose between the same antibody sold under different names.  If all else is equal, feel free to make...

...expression of kynurenine aminotransferases, to enhance the conversion of kynurenine into kynurenic acid. That...skeletal muscles led to another clue: differences in the kynurenine pathway of tryptophan degradation. It...That’s key because, unlike kynurenine, kynurenic acid can’t cross the blood-brain barrier. By lowering...plasmids from Addgene. References: Skeletal muscle PGC-1α1 modulates kynurenine metabolism and mediates...lowering circulating levels of kynurenine in the body, the brain is protected from stress-induced biochemical...

...’s of working in different sectors. Luckily, I have worked for a variety of different organizations, so...overview of my experience and how I felt about the different positions to enable them to decide for themselves...effectively. I had to learn how to cope with a different climate and culture, and also needed to be resourceful... experience and a second language in Spanish or French, and so they were ideal candidates for these positions...very quickly; as soon as I learned how to do the different tests and became efficient, I then started to ...economy and plenty of jobs in science at world renowned institutes. The lab at the University of Queensland...diagnostic tool for the detection and diagnosis of different types of pancreatic cancer and highlighting which...

... and reference on how to design PCR primers.  Types of PCR Since the invention of PCR, different PCR methods...Analysis RNA detection     References and Resources References Shen C-H (2019) Amplification of... methods have been developed for different scientific applications. These PCR methods all use the same...referred to as simply PCR, is used for an array of different molecular biology techniques and experiments. ...alleles or mutations. ddPCR also does not require a reference or standard curve, which can be time consuming...original template DNA in the sample.  Addgene currently uses ddPCR for AAV titrating. You can learn all...Multiple template PCR - multiple templates with different primer pairs that align to the target region of...

...genetically modify primary cells. Amongst the different vectors used, lentiviral vector constructs have...CaCl2, cationic liposome) or electrical (electric current forming membrane pores) transfection. These techniques...transducing certain quiescent or terminally differentiated cells such as macrophages and microglia. This...ability to infect non-dividing cells. Thus three different generations of lentiviral vectors have been established...transcribe the viral genome in producing cells. Currently, the third-generation lentiviral vector system...to RCL. Regarding transduction efficacy, no differences between the 2nd and the 3rd generation of vectors...marker can be placed under the control of two different promoters. Both the single and dual promoter transfer...

...about your different experiences in research labs. What are some similarities and differences you have ...found that research institutes in different places have very different cultures. In Basel, I felt that ...developing drugs against intractable diseases. Currently, a number of labs are using enChIP, and I'm very... a wide variety of biological systems. We are currently developing several additional systems of enChIP...recently developed the oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) method to suppress PCR amplification...world? Fujii: Fortunately, I was able to work in different parts of the world in my early research career...which is not trivial, and the grant situation is currently much better than that in the US for me. In addition...

...insensitivity to other ions In BRET, A mutant Renilla luciferase, catalyzes the oxidation of its substrate...applications. The system can be readily modified to use different antibiotic resistances, restriction enzymes and...either without a tag or as multiple fusions with different N- and C-terminal tags. Additionally, by using... a final lentiviral vector containing up to 5 different elements. Lentiviral vectors constructed with ...instance, the intra-nuclear distance between loci on different chromosomes. The advantage of having this kind...targeted loci during the cell cycle or cell differentiation. This tool should delight anyone interested...function. In order to dissect protein traffic in different systems, Franck Perez’s lab, from the Institut...

... pivotal role in spontaneous swimming. References Arrenberg, Aristides B., Filippo Del Bene, and Herwig...Optogenetics in a transparent animal: circuit function in the larval zebrafish." Current opinion in neurobiology...wavelengths and light intensity requirements of different opsins, studies have shown that some channels,...simultaneously and still be individually activated with different lights (Baier et al., 2009).  Zebrafish behaviors... do so, the lab expressed the LiGluR opsin in different subsets of spinal cord neurons using the Gal4/...Gal4/UAS system. This created fish lines with different groups of neurons that could be activated with light...spontaneous swim or escape, was elicited when these different neuron groups were activated or “turned on.” In...

...number of different experimental approaches. They first assessed the activation of different members of...response to different agonists. These studies showed the promiscuity of GPCRs for different G protein transducers...neurotransmission. But with approximately 950 different human genes encoding GPCRs (Takeda S et al., 2002...a vast number of signaling cascades, inducing different cellular responses. One of the biggest advantages...measure the physical association of combinations of different Gα, Gβ, and Gγ subunits. When inactive, these ...is used to excite an acceptor fluorophore. The difference for BRET is that the energy transfer is between... the signaling through the transducerome from different GPCRs. This collection can be used to study endogenous...

...reprogramming factors and wait for cells to de-differentiate. However it’s difficult, especially as a newbie...reprogramming proteins. Table 1: Key Features of Different Methods for Generating iPSCs. One important note...reactivation of Myc or other oncogenes after differentiation has been linked to tumor formation in iPSC-...concerns, but overall integrating viral systems currently lack the safety required for translational use...iPSC-derived cells can interfere with their differentiation potential. Finally, retroviruses are prone ...drawback is that lentiviral vectors insert in different genetic locations in each transduced cell, so ...a single polycistronic transgene or with four different adenoviruses, each expressing one factor. These...

... about the current state of a partnership. Every collaboration and partnership is different as are the...collaborators from other institutions personally on conference calls and make sure to maintain these relationships... learning from others and meeting people with different mindsets and approaches can truly broaden your...neighboring lab, you've met interesting people at conferences and you have successfully reached out to people...collaborations and partnerships. There are many different ways to maintain a relationship - many of them...chat can be rather tricky (think people based in different countries). Here social media is a connector and...maintain external collaborations is important? In my current job at Addgene I work with people from various ...

...lead to wildly different ideas of how to approach a problem, but it is these very differences that make our...similar cognitive biases and this would become apparent in the software we develop. We would all have ...the software design. Each of us brings slightly different cognitive biases as a result of our diversity ...Another advantage of Scrum is that it provides transparency to everyone involved about the status of each...Addgene and ourselves. We are also able to consider different technical approaches that may come up during discussion...developer and I get to see an approach that is likely different from what I would have done. And finally the stakeholder...isn’t how we work. We all take turns working on different parts of the code. I think the best way to learn...

... that can replicate in different bacteria and express proteins within different host organisms. pBBR1-...recombinant vectors at Addgene!   References and resources References Doran, K. S., Konieczny, I., & Helinski...  A plasmid’s host range is determined by the different hosts that a plasmid can transfer to, replicate... they carry among organisms belonging to many different phylogenetic groups (Jain and Srivastava 2013)...plasmids are more stable, can be easily cloned in different bacteria, and can be transferred to various experimental...and can alter the efficiency of replication in different bacterial hosts (Doran et al. 1998). Regulatory...plasmid has multiple ori that are functionally differentiated from one another. Based on their structural...

...not fluoresce. A dark population is not readily apparent and can confound quantitative imaging experiments...posttranslational modifications can disrupt FP folding rendering the protein non-fluorescent. Unfortunately, accumulated...palette of FPs (moxFPs) [5]. The moxFPs overcome the inherent problems of popular fluorescent proteins and were... green and blue or cyan and yellow moxFPs. The inherent brightness and spectral characteristics of the...the moxFPs are comparable to the non-optimized parental proteins. Our recent publication illustrated the...most, if not all, of the FP fusions. Our moxFPs currently represent one of the most inert solutions for ...at the National Institutes of Health and he is currently an Associate Professor at Albert Einstein College...

...multiple times and on different days. Using our example above, we would test different preparations of Plasmid...Plasmid A in different aliquots of cells on several different days. Biological replicates control for ...There are many, many different types of experiments carried out by scientists every day. Although the... the results because it provides an important reference point when analyzing the specificity of the shRNA...variable should be consistent over time and from different preparations of the purified test plasmid expressing...a bit more below, but the papers cited in the reference section provide more in depth information. Technical...aliquots of cells and plasmids, be performed on different days, etc, but outside constraints may impose ...

...endoderm progenitors (iEPs), a self-renewing cell type that can differentiate into hepatic or intestinal cell... tool. Check out the CellTag webpage! References Biddy, Brent A., et al. "Single-cell mapping of lineage...Direct reprogramming describes the process where differentiated cells are turned into a cell type of choice...identifier and allows cells to be tagged with different CellTag libraries at multiple timepoints during...inefficient. Cells were sequentially tagged with different CellTag libraries at three time points during ...are undergoing identity changes, such as the differentiation of induced pluripotent stem cells or the clonal...reprogramming and direct reprogramming Explore different methods for delivery of reprogramming factors ...

...of iGluSnFR   iGluSnFR was developed in 2013 by Loren Looger’s lab at HHMI Janelia Research Campus to ...medium affinity for glutamate), that came in three different colors – blue (SF-Azurite-iGluSnFR), green (SF-iGluSnFR...glutamate signaling events, and accurately differentiating between synaptic vs. extrasynaptic glutamate...and ∆F/F0. Introduce the two variants in three different membrane-targeting flavors – PDGFR, GPI, and SGZ...Figure 1   Increased versatility through two different variants The yellow (v82) and lime green (v857... Figure 2: Glutamate titration curves for the different iGluSnFR variants. Image adapted from Aggarwal...Figure 3: pH titration curves and ∆F/F0 for the different iGluSnFR variants. Image adapted from Aggarwal...

...duration of the conference (be warned, the hand sanitizer does go quickly!)  The Conference How many Addgenies... to brush up on your conference skills, check out some of our helpful conference blog posts before you...Scientific Conference Poster Session TipsHow to Make Friends and Meet People at a Scientific Conference New ...Getting ready for Society for Neuroscience Conference 2023? So are we! Come say hi to us at Addgene's...favorite content strategist, Jo. While we've all got different areas of expertise, we are each equally excited...invite you to join us! We'll also be sharing our conference experience on X, Instagram, and Bluesky.   Finally...

...between Cas9 enzymes from different species, explaining why they recognize different guide RNAs. Deep in the...Other CRISPR enzymes like Cas12 have different structures and different uses. In all cases, structural biology...includes most of the bridge helix. Created with BioRender.com.   Cas9 has two major lobes, called NUC ...guide RNA (PDB ID 4ZT0), shown opaque (left) and transparent (right). The RNA guide sequence is extended as...target DNA (PDB ID 7S4X), shown opaque (left) and transparent (right). Some nucleotides of the non-target DNA...from the same angle to highlight conformational differences. HNH, red; RuvC, orange; CTD, yellow; REC lobe...; DNA, gray; PAM site, yellow. Created with BioRender.com.   The exact conformational changes that...

...findability” of the information on the site. Different users have different strategies for finding what they need...the overall user experience of our website, the different website navigation paths, the organization and...software team tailored Elasticsearch to support the different usage patterns the larger team identified. The...our catalog of materials and services is quite different from the way it searches through our educational...information in our search indices in order to support different kinds of matches: exact words, partial words, ...or numerical identifiers. We also combine the different terms in a query in a way that allows users to...Weighting We determined the relative importance of different bits of information for a given resource, and ...

... thinking, ‘wait, those terms mean different things?!’, you aren’t alone! We’ve noticed a lot of scientists...in. The differences They say the devil is in the details - and that’s where the differences in these... RNAi resources and go fishing! References and resources References Wang, T., Wei, J.J., Sabatini, D....selections The common goals The confusion in differentiating between a screen and a selection isn’t surprising... you would individually treat cells with many different drugs and then perform an assay to look at an ... No one wants to spend time screening hits that aren’t “real” or novel for their purposes… or worse yet...experimental question. Now that you understand the difference between the two, it’s time to check out our CRISPR...

...overexpress or knockdown a gene, you might need different plasmids. Harvard’s DRSC/TRiP Functional Genomics...varying expression levels of your transgene in different fly lines. Also, if there is a chance you need...transgenes in one fly, having the transgenes in different loci allows for genetic recombination so that ...for direct comparisons between lines expressing different variants of YFG because the transgenes are found...remember that you’ll need to integrate them all at different loci. Fortunately, there are many docking sites...docking site is located). This amounts to a small difference of two or three weeks. 3. How would you like ...experimentation. Drosophila are amenable to many different kinds of experimental assays depending on your...

...containing many different promoters, RBS’, coding sequences (CDS), and terminators of varying strengths. These ... developed for the study of enhancer function, strength, and binding (e.g. DHS-seq, FAIRE-seq, and CHIP-seq...variable availability of enhancer sequences in different chromatin states. Alexander Stark and colleagues...transcription from the minimal promoter and its strength can be determined from its enrichment over input...process of cloning, combining, and testing these different parts can be quite arduous. The cloning process...that control many cellular processes such as differentiation, proliferation, and apoptosis. In humans, mutations...To facilitate innovative cancer research, the Reference Reagents Group of the NCI RAS Initiative, led ...