...viral particles that can transduce cells, which can vary based on target cells and can be altered by freeze... where you can obtain excellent quality, high titer rAAV (including Addgene!), but you can also generate...due to their high density, which can be collected afterwards. You can then remove the density gradient...system, HEK293T cells, expressing Ad E1A and E1B, can be transfected with your transfer plasmid, carrying...titration by qPCR.   Pro tip! An optional media exchange can be performed 12–18 hours post-transfection to help...For certain in vitro and in vivo applications, you can consider using unpurified AAV from the supernatant...Purification To purify your rAAV from your supernatant, you can simply use polyethylene glycol (PEG). We add 25 mL...

...ratios with such techniques can be problematic, however. Well designed aptamers can help reduce such background...temperatures, since proteins can denature while aptamers are more stable and can go through cycles of denaturation...bacteria). That range is possible because aptamers can either fold to incorporate their target molecule,...enzymes, such as: Design and selection for targets can be done largely in vitro; Easy and reproducible ...antibodies for diagnostics know how frustrating it can be to get a “bad batch”); Customizable sensitivity...aptamers glow The flexibility of aptamers means they can be used with or as fluorophores in several different...aptabeacons (or aptatags) were born. Aptabeacons can be created in many different ways, but the general...

...and analysis.  As a rule of thumb, you can assume that qPCR can accurately detect expression differences...differences, and it can even be used to detect a single copy of a transcript. But this sensitivity can sometimes...configurations can help you identify pipetting or evaporation errors. And if time allows, you can fit in a ...statistical significance (Taylor, 2017). With the right conditions and parameters, some people find it can detect... requires significant optimization to ensure the results are correct. Primers and probes can interfere...troublesome, multiplexing your reference gene set can save significant plate space and may be worth the initial...to measure relative quantification of mRNA, which can be used as a proxy measure for differences in protein...

...in non-specific staining can be a challenge in the best of cases. But it can be particularly challenging...(Figure 2B) Some labs have found that you can significantly decrease background staining by mixing your...same levels of endogenous IgGs, so some researchers can get away with species on species staining with no...    Figure 1: Species on species staining can be a problem because the secondary antibody is not... However, directly conjugated primary antibodies can be expensive and may not provide enough signal to...1998). After washing away unbound fragments, you can then apply your primary and secondary antibodies ... Proper tissue processing and sample preparation can also help reduce the levels of endogenous IgGs in...

... fun example and you can find it in our Mentoring for Scientists Guide.  You can use it to clarify and...step you can take is to set goals to work on together and track progress at every meeting. It can take many...impart. An undergraduate can mentor a high school student, a graduate student can mentor an undergraduate...Series. The entire series and additional resources can be downloaded in E-Book format at the end of this...setting to your mentoring relationships, so that you can reap rewards in the form of reaching career development...undergraduate, a postdoc can mentor a graduate student – you get the idea. There are many advantages to being a ... Schedule meetings or calls regularly and don’t cancel, so that you drive progress with regular accountability...

...scientist. In this blog, we’ll delve into strategies you can use to foster innovation. If you identify as a non-creative...yourself a block of time and a quiet space where you can address the issue head on. Prepare for this time ...environment where everyone has their ideas heard.  So how can we assure that all participants feel comfortable ...devil’s advocate is elected, others may feel they can share opposing opinions as well. I am sure we have...goal is to generate unique ideas. Recognize ideas can come any time. Meetings shouldn’t be the only place...ideas are not well received, understand that people can be resistant to change. Ask your peers or your superiors...fixated with the idea of making something the best it can be that it seems impossible to start at all. This...

...the best speaker you can be. Seek out opportunities to practice and over time you can master this important...solutions—public speaking is skill that any scientist can learn.  Any time you give a science talk, you are... added bonus to knowing your audience is that it can help you avoid making mistakes. I once made a joke...a narrative connecting cause and effect. Stories can and should be very simple. The goal is to get the...to include that material, but don’t force it. You can always relate to scientists by explaining how your... real audience Good, even great, speaking skills can be attained by almost anyone with enough practice...previous year. Usually fewer than 10% have done so. How can you expect to deliver a great public presentation...

...are looking, you can either scan the genome yourself for potential gRNA sites, or you can utilise several... or you can order them yourself through whatever DNA supply company you currently use. You can also find...sequencing comes back. Alternatively, if you can afford to wait, you can hold off on genotyping until the pups...targets in that region. The CRISPR Software Matchmaker can be used to determine the pros and cons of using many...genomic fragment containing the sgRNA target sequence can be placed in multi-cloning site (MCS) between EGFP... and sgRNA expression cassette. The target sgRNA can be cloned directionally into the BbsI site. (c) The...sequence surrounding your gRNA. Once amplified, you can insert this region into a pCAG-EGXXFP plasmid using...

...out to your peers and other mentors who can help you. You Can Talk to Your Editors I wish I had been... was “How many graduate students/post-docs?” You can find some of the more detailed questions the organizers...chosen career path,” but individual institutions can have many different names for the positions commonly...universities have simple postdoc numbers sorted out there can be gaps in knowledge of demographics and the information...environments. Slide from Paula Stephan’s Talk (All slides can be found here). Importantly, the panel offered some... panel, focused on showing researchers that they can take steps to achieve success in their careers. The...the coming months and welcome guest bloggers who can provide additional advice to trainees navigating ...

...sequences determines the orientation of the ITR, which can either be “flip” or “flop” (Figure 2). Additionally... elements of AAV and rAAV vectors, modifications can drastically affect gene transfer efficiency and transgene...important for viral genome replication, these mutations can affect transduction efficiency and subsequent transgene...sequence containing transcription factor binding sites can increase transgene expression and transduction efficiency...efficiency (Shitik et al. 2023). Transgene expression can be altered by using ITR sequences from different ...activity (Earley et al. 2020), but non-AAV2 ITRs can be used to drive gene expression at varying capacities...capacities. AAV vectors containing the AAV2 ITRs can be cross-packaged with capsids from multiple AAV serotypes...

...that can happen is someone will say no, but if you don't ask, you will never get the help that can make...the most effective tactic a scientist in training can adopt to ensure opportunities in the future. Studies...relationships as part of the job application process can dramatically increase the chances of being seriously... submitted The structure and content of a resume can be crucial for a successful application. It takes...good at learning new things—this is a strength you can draw on and demonstrate in your application). Beware...During the entire process, use whatever means you can to get your resume into the hands of the hiring manager...great way to find an appropriate liaison, but you can use alumni databases, friends, and even relatives...

...needed, a layer can quickly be removed and disposed of without risking exposure. You can request disposable...meaning it can’t replicate and continue the infection cycle once inside the host. While the virus can still... to integrate into proto-oncogenes, which can lead to cancer. These viruses need to be handled in a BSL...useful because they’re derived from viruses that can infect people, cells, and animals. Some of these ...these viruses are completely inert, but others can cause diseases. Nervous about handling viruses? That’s ...to take, and if something does happen, ensure you can receive appropriate treatment and/or monitoring. ...as part of the viral infection process (though it can integrate at chromosome 19q13.4 qtr. (AAVS1)). For...

...examination of specific proteins within a system. This can be pretty complicated given that a single cell has... purification. In this strategy a single protein can be isolated from a complex mix using affinity chromatography... tag, on the protein. While a variety of methods can be used to successfully isolate proteins, this blog...metal ions such as Ni2+ or Co2+ (Hochuli 1988), and can elute proteins with up to 95% purity (Janknecht 1991...histidines are present in most production systems and can bind and co-elute with the protein of interest (Bornhorst...denaturants (Hochuli, 1988). In some cases, proteins can be bound under denaturing conditions to expose the...preferable over the other however, tag placement can affect protein function. B, The string of histidines...

...probes. You can follow him on twitter: @joachimgoedhart.   Marieke Mastop is a PhD candidate at the Section... best fluorescent proteins for live cell imaging can be excited many times, thereby producing a large ...power is a property of the fluorescent protein. This can differ between fluorescent proteins in the same spectral..., the photostability of the fluorescent proteins can be directly compared. An example of a photostability...distribution. Therefore, photostability measurements can be performed with soluble fluorescent proteins or...make these measurements and hope this information can help guide your understanding of reported data. Photobleaching...loss of fluorescence, which, in its simplest form, can be described by a mono-exponential decay (analogous...

...markers can be challenging! But we hope this guide can help you get started. Remember, you can always ...This can be helpful as you're learning! Ashlyn has a research background in CD8+ T cells and cancer immunotherapies...By compensating between fluorescent channels, you can correct for this spillover and more easily separate...your samples. This means that, unlike voltages, you can adjust compensation values later in your analysis...for APC (Figure 1B). For single-color samples, you can only change the compensation values for whichever....     In general, most spillover between samples can be fixed with fairly low compensation values (0-50...machines or the program you’re using, but usually you can click on the correct box and either manually change...

...stock, while you can use the remaining vial to grow and freeze additional cells that can be used as your...UV light 3-4 times a week for up to half an hour can help keep your hood contamination free, but you should...disinfectant (most people use 70% ethanol, but you can also use Incidin wipes) before and after your work...materials you’ll need to bring into the hood and can wipe them all down before you start. This will also... difficult to clean your workspace properly, but can also disrupt the laminar air flow around your work...have a light microscope close to the hood so you can view your cells regularly (both before entering the... you’re working with so that cross-contamination can be identified. Even if some mammalian cells lines...

...soul of Addgene’s mission), but legal complexities can arise when scientists are sharing their materials...publications, and commercial use often come up. An MTA can resolve those questions! MTAs protect you and your...all those benefits, we know that legal documents can get tricky. Here’s how Addgene helps: For Providers...distribute your materials under the UBMTA terms, so you can rest easy knowing your rights – and the rights of...generates the MTA and emails it straight to you, or we can send it directly to your institution’s MTA coordinator...Organizations that have opted into our eMTA system can even sign electronically or choose to auto-approve...result in quick approvals, so your plasmid requests can get to your lab faster. For Everybody: Friendly...

...performed at Addgene, this adds up to a significant time savings. You can also save yourself time if you're ...step. Read on to find protocols and tips that you can use in your own lab. How to make your own competent...competent cells because cells prepared using this kit can be transformed without heat shock. Detailed protocols...cells using the protocols provided above, then you can find the subsequent transformation protocols below... Cells prepared using the Z-competent TM buffer can be easily transformed without a heat-shock step. ...resistance gene time to be expressed.  However, you can skip this incubation step and go straight to plating...other competent cells) to ensure that your cells can be readily transformed. Compare the number of colonies...

..., 2020, 2022). I do mean highly multiplexed: you can image over 65 different parameters using this method... from Radtke et al., 2020). This flexible system can be used with many conventional microscopes, amplified...that they used in their experiments. Researchers can then visit the IBEX imaging community discussion ...conversations with other users. The entire dataset can be downloaded from Zenodo, including the reagent ...for easy filtering and sorting. Reference images can be viewed by clicking on the links in the "Agree"...detergent, antigen retrieval conditions, and more. Users can also recommend or not recommend a particular reagent...discussion forum where scientists from around the world can ask and answer questions. Andrea and Ziv were...

... reach. We can’t save you the hard work of reading papers and doing experiments but we can definitely ...large section of open access life sciences data. You can use ReFigure to coherently save analyses with the...insights on a topic. Beyond collating other’s data, you can even include your own data in a ReFigure. For example...Figshare is currently supported by ReFigure beta) you can include it in a ReFigure. ReFigures are good for ...proposals. ReFigures are one such research product which can convince reviewers of your expertise and insight....minutes, visit our YouTube Channel. Currently we can only connect figures from PubMed Central, eLife, ...Download ReFigure extension and visit ReFigure.org. You can reach the ReFigure team at [email protected] ...