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...will turn yellow. Do NOT change the media (add 2-3 mL of fresh media once a week), and do NOT harvest the...transgenes, transduce quiescent and dividing cells, and do not integrate into the host’s genome. The main challenge...test for the presence of RCA in the viral stock by doing a plaque formation assay on A549 cells . Alternatively...at -80C. Purification and concentration kits that do not use CsCl are commercially available. Titer calculation...

...such as HEK and CHO do not seem to require this, while nearly all endocrine cells do. While not all of ...different densities, then dispose of the plates that do not appear to be yielding well-spaced clones. For...replicates of each clone in case of contamination. We do not normally use antibiotics. Lastly, and perhaps...experience, cell clones picked using the agarose method do not differ grossly from those isolated using cloning...

.... In the most practical terms, what GreenGate can do is “save you half a year,” in comparison to more ...sensitive protein,” he says. “There is nothing you can do about it but be gentle. If it no longer works, just...blocks will be available, making it easier for us to do our work.” References 1. Athanasios Lampropoulos...

...purification you can do is a miniprep, but if you need larger quantities of DNA, you’ll need to do a midiprep or...systems. Read on for an in-depth breakdown of how to do perform restriction digests.   Overview of...any pieces cut out or off of them. An easy way to do this is gel purification. In gel purification, you...empty plasmid, you will still have colonies when you do not add ligase. If the colonies are a result of recipient...significantly more colonies when you add ligase. If you do not see any colonies, you should conduct a positive...

... better equipped than anyone else in the world to do it.  In the lab, it might mean persuasively articulating...should change established theory. So, what can you do to ensure that your skills in science communication...presentation slides, learn from audiences as they do science outreach and engage with the public, and ...science communication or start your own! When you do, let us know!  To get in touch with ComSciCon, find...

...central We’re working on a project where we aim to do large scale DNA preps with multiple samples without... libraries as opposed to replicating them like we do at Addgene), going to the workshop was a great opportunity...What are the familiar parts of this workshop? What do I understand already? What don’t I understand? How...experience in my first blog post! I highly recommend doing the same if you ever go to a workshop because it...

...to see if the type of science we do fits in with what they want to do. The experiments must fit with their...strive for a common big-picture goal. It is worth doing some some serious pre-work to find a lab that will...is this PI looking for in a new researcher?,” “How do I convince the PI to choose me?” We asked three leading...

...Although plasmids do not naturally exist in mammals, scientists can still reap the benefits of plasmid-based...transfection. Unlike bacterial transformation, scientsts do not "select" for cells that have taken up the plasmid...concentration for selection in mammalian cells. Before doing a transfection experiment, it is important to determine... and is not appropriate for mammalian selection – do not confuse this with G418 (aka Geneticin). Neomycin...

... are an expert networker (or if you just aren’t doing it at all yet) watch my webinar, “Not Networking...constructive suggestions and feedback. The best thing to do to make your mentor want to keep working with you...This was really helpful, perhaps you wouldn’t mind doing it again next month?” Some mentoring relationships...last for an hour or for a year. Some click and some do not. These are all normal outcomes. If you have a...

...Producing Your rAAV AAV Titers: Where Do They Come From and What Do They Mean? Overcoming the Challenges...viral production, particularly for labs that already do mammalian cell culture. HEK-293T cells are the most...application. With mammalian systems, it is standard to do only one passage (transfection). This differs from...and widely available. Unlike mammalian cells, they do not require serum or an incubator to grow. The virus...

...What do a viral vector production facility, food allergy testing lab, and the grad student down the hall...concentration of a target molecule in an unknown sample. To do this, you serially dilute a control sample of known...determine the concentration of the unknown sample.  How do I make the standards for a standard curve? You create...somewhere in the middle of the standard curve. If you do not know what concentration to expect, you may need... should be within 15% of the expected value. How do I plot and use a standard curve? To create a standard...

...’s not always easy to figure out what you want to do after graduate school, at least not while you’re ...your career goals, find people who are. I wanted to do fun science activities with the public, so I reached... interests! Especially as someone interested in doing science outreach, not only can I easily share science...graduate students because I wasn’t sure what I wanted to do. A few weeks into volunteering, I was talking to ...said, “Boy, I love science,” and I realized, I still do too! Beyond the outreach experience I gained from...

... Organizing your lab notebooks, protocols, and to do lists can be easier than you think! Here are three...spot the colors and text. Tip 2: Use electronic to-do lists to access your tasks on the go Never miss a...sync everything to your calendar. Ditch the paper To Do List and move to one that moves with you. Use software...

...types and deliver any cargo you fancy (size limits do apply, however.) Why use a virus? Viruses are essentially...viruses Adenoviruses infect non-dividing cells but they do not permanently integrate into the host genome (non-integrating...(non-integrating) as lenti and retro viruses do. They are generally used to achieve transient, high expression...adenovirus is their low immunogenicity, which means they do not stimulate a strong immune response when introduced...

...transduction coverage compared to just one vector.   Q2: Do you think it is possible to make an efficient AAV...method to be reliable and reproducible. However, we do not use amplicons in ITR as we have found this to...mice, e.g. first at 2 months, then at 4 months? Or do antibodies become a prohibitive problem? A: Re-administration... suppression to allow AAV re-administration but I do not think the field has settled on ‘best practices...

... thing you’ll want to do is store it away safely. The first thing you should do is record the manufacturer...accidentally leave your antibody out on the bench overnight. Do not panic! Typically, antibodies are okay if they...however, is pretty much every other assay you can do. Most non-HRP conjugates are fluorophores, which ...

...in orange. Created with BioRender.com.    How do secondary antibodies work?  Secondary antibodies ... such as the rabbit IgG class of antibodies. They do so by targeting the Fc region of an antibody, the...But in reality, multiple primary antibodies can and do attach to a single target protein, so this assumption...exception of flow cytometry, most immunoimaging assays do not allow for absolute quantitative analysis of a...

... is not far enough along, scientifically, to be doing in humans; We have not had a broad, public conversation...reproduction, the relevant document for the question of “how do we move forward” is the June 2012 report on the ethics...ethically fraught rapidly emerging technology. We could do this for CRISPR/Cas9 – or any other germline editing...adequately proven to be reasonably safe and effective. We do that through a standardized process of good research...

...while some might argue that the only way to do something is to do it huge, there are times when a more incremental...phenotypes, such as cell-non-autonomous effects, that do not lend themselves to screening in a pooled setting...

...with fluorophores whose excitation/emission spectra do not overlap. Multiplexed IF can also require some...the performance of other antibodies. But, once you do find the right protocol, multiplex IF can give you...use and the ultimate outcome of your assay. So how do you choose good ones? Choosing a secondary is often...available imaging instruments and the spectral properties do not overlap with any other fluorescent molecules ...antibody will likely perform as expected in both.  How do you find validated antibodies? Many companies that...