...protein expression varies from sample to sample. To compare one sample to another, you’ll need to normalize...curve, your standard, and your sample(s), as you can only compare samples to a standard curve generated...published antibodies, check the manuscript and supplementary figures for validation data, or follow the references...western blot. Polyclonal antibodies, which bind to multiple sites on an antigen, are cheap and can help amplify...overnight or 1–2 hours at room temperature (RT). Most people choose to incubate their primary antibody overnight... likely higher background noise as well, and multiplexing is challenging and limited. Direct westerns ...conjugated to primary antibodies). Direct westerns are simpler and faster than indirect ones and can be used in...

...place people share ideas. Ideas had over lunch are just as valuable! In 2019, Addgene implemented a company-wide...funding in the world? What if you could hire more people? What would you do with unlimited time? Another...brainstorm session is to start with something completely unrelated to the issue and consider how it could...expert at Stanford conducted an experiment in which people were asked to create a device to help students ...avoiding groupthink, the tendency of a group of people to agree with the most prominent idea, and instead...comfortable sharing their ideas? And how do we give people the opportunity to speak out and prevent groupthink...you meet. This holds the dual purpose of giving people time to process the issue properly while also discouraging...

...choosing antibodies   Complexity is a key factor Biological systems tend to be complex with a variety of ...Experiments tend to be equally complex with users trying to probe multiple targets in parallel. In these...antibody) approach?  Staining for multiple targets When staining multiple targets indirectly, you must strategically...If you are lucky in lab life, you will have a plethora of antibody options for your experiment (all well...Some secondary antibodies are broad and recognize multiple isotypes from a species, like a general anti-mouse...than a broadly  reactive secondary (Fig. 1). For example, an anti-mouse IgG2a secondary would be a better... rabbit versus mouse monoclonal antibodies for example. Rabbit monoclonal antibodies have higher affinity...

...flat-bottomed plate. The basic principle of an ELISA is that any antigen within a sample adheres to the well (either...wells with an experimental sample allows for detecting one protein from the sample. A direct ELISA has many...require scientists to detect proteins in their samples. One technique that relies heavily on antibodies...antibodies coating the wells to capture antigen from a sample, whereas antigen is bound directly to the wells...protein you are interested in detecting in your sample. When an ELISA kit is ordered, the wells are typically...already coated with the antibody. Then, you add your sample to the wells, and the antibodies bind to your protein...ELISA is best used to detect proteins for which multiple antibodies that recognize different epitopes are...

...Perfect Science Talk." 4. The chalk talk, simple in principle The first question I usually get about chalk...relate to, that people would agree is important to understand. (1-2 sentences). For example: Distinguishing...you have any questions, comments or suggestions, please do not hesitate to email me at [email protected]’ve provided guidance and mentoring to several people that found my insights helpful. All went on to ... need a Nature/Cell/Science paper. You do need multiple first/corresponding author publications in quality...for feedback), possibly the Dean, and even some people in your field that still need to understand your...slide deck, markers, etc. Chalk or markers force people to be more succinct. Slide decks leads to the opposite...

...individual experiment. Practical example: lymphocytes in a mouse tumor sample You made it! So much about the...When using flow cytometry to analyze your samples, it is necessary to set up a sequence of gates to be...working with a heterogeneous cell population, for example from a processed piece of tissue, where cells are...variations in those factors. To understand the principles of gating, you’ll first need to learn a little...size, while SSC values depend on the structural complexity inside the cell or on its surface. Bear in mind...on the cell’s size, morphology, and structural complexity.   Height, Width, and Area Within FSC and...the area under the resulting curve. By default, a sample on the flow cytometer (before you set the first...

...important” or senior people. Focus on building relationships with peers and people around you naturally...run. Don’t worry about the people that don’t respond; focus on the nice people who do respond. Don’t call...connecting with people you don’t like or who do not show interest in you. Network with the people that you want... it’s about building genuine relationships with people you care about knowing. How do you follow up with...the encounter. As a side note, my guess is that people will not be exchanging business cards for awhile... you have something to ask or say. It can be as simple as “it was nice meeting you.” Include follow-up...who helped you succeed in any way. Don’t believe people who tell you that thank you isn’t necessary. I ...

...expediently join multiple synthetic fragments, create multiple mutations in one or multiple fragments, and...joining multiple fragments of DNA to create a synthetically designed DNA sequence. There are multiple methods...becomes as simple as mixing fragments together with linearized vector for seamless joining of multiple mutations...standardized constructs and tools for the rational and simple shuffling of DNA regions. Golden Gate Assembly ...Assembly Tool which is freely available online. Its simple interface can guide you through both experimental...Assembly Mix include:  Generation of constructs with multiple point mutations Cloning sgRNAs Production of Yeast...Expression Cassettes Generating constructs with multiple point mutations NEBuilder HiFi can simplify multi-position...

...and so it may be necessary to try multiple techniques. For example, we have found that some antibodies...detecting the target-of-interest in your assay and sample-of-interest. The approaches available broadly map...determining the selectivity of antibodies for other sample types and for non-surface (intracellular) antigens...protocol used and the abundance of the epitope in the sample, relative to cross-reactive antigens – that is,...approaches used will be different depending on the sample and target type. To start the validation process...antibody validation, and it is recommended to screen multiple candidate antibodies when possible. If cost is...the target at a robust level, similar to that in samples of interest. If you are working with cells that...

...success or failure: In our second example we’ve added some simple but critical logic. Some steps fail... a new tube. In the first example we’ve used inheritance to tightly couple the contents of a tube with...appropriate glycerol stock within our freezers Pick the sample into the appropriate stab culture Grow the stab...proper location To keep ourselves from mixing up samples and to ensure that everything is grown successfully...needs to handle these cases. The following code examples show how software can be structured to allow for... for the messiness of biology. These examples are intentionally trivial, but they're here to help tell...tell this story. In this first example to the right (# this will make you sad),  someone has put in a request...

...compatible with multiple FP(11) fragments, even from a different base FP (for example, EBFP2(1-10) can...generally known as Bimolecular Fluorescence Complementation (BiFC). It can provide a qualitative or quantitative... or even single-particle tracking of the bound complex. There are so many FPs available, with many ways...because they are used with an already-available complementary fragment. Next, let's look at a round-up of...EBFP2(1-10) and Capri(1-10) for use with GFP(11) Multiplexed labeling of cellular proteins with split fluorescent...Cerulean Cerulean(1-10) for use with GFP(11) Multiplexed labeling of cellular proteins with split fluorescent...protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. Shyu...

...DNA strands complementary and non-complementary to the gRNA, respectively. When complexed with a truncated...full-length gRNA (20nt complementarity to target site) or truncated gRNA (≤15nt complementarity to target site...with which it is complexed rather than the protein itself. When native Cas9 is complexed with a gRNA that...transcriptional activator to the Cas9-gRNA complex. When this complex is directed to a target upstream of the...bioengineering, as it has facilitated the development of a simple and scalable means of making targeted genetic edits... directed to virtually any genetic locus when complexed with an appropriately designed small RNA, or guide...conventionally contains a 20-nucleotide sequence that is complementary to the target site, or protospacer, in the genome...

...by expressing multiple crRNAs in parallel (Fig. 3), multiplexed insertions into multiple distinct targets... The requirement for multiple Cas proteins to form the Cascade targeting complex is a hallmark of Type...nuclease activity. Instead, it forms a novel co-complex with the transposition protein TniQ for targeting... by the system. Guided by the crRNA, QCascade complexes survey the genome and bind target DNA sequences...pairing. Separately, TnsABC forms a transposase complex with the mini-transposon, excises it from the donor... report of VchINT, three separate plasmids and multiple promoters were used to express the necessary components...in single-plasmid INTEGRATE (pSPIN), which offer simple transformations for smaller cargos, and the ability...

...group and topic. For example, it may be useful to adapt the second version (multiple presenter, figure by...time that accommodates more people. The feedback could be solicited via multiple avenues including casual...in journal clubs. They also have likely had the pleasure of attending both productive and not so productive...and maintain. It became clear that while several people across teams had various levels of experience with...decisions to make. How often will you hold it? How many people do you expect to attend? What is the expectation...ensure success. One of the most important is letting people know it’s happening. Aim for 3-4 weeks advance ...advance notice for the first meeting so that people can set aside time in their schedules and read the paper...

... pros of multiple channels/balance out cons. Flexible. Can allow people to learn in multiple ways from... means. Easy to combine multiple types of media. High barrier to entry (people have to enter a building... often unintentionally plays a big role in what people think of as "the best way" to communicate their...work just as well for everyone else?  In truth, people are incredibly varied in how they prefer to consume...However you choose to communicate your science, some people will love it and others will avoid it like the ...socioeconomic status and geographic location. For example, not all social media platforms are available or...probably didn't sound like a great idea to most people in 2005, but XKCD is pretty successful!  What resources...

...barcodes for live cell clonal multiplexing by: Rachel Leeson Multiplexed images in live cells remains ...own subclone, they were able to develop a 12-plex multiplex imaging system. Mixing the subclones within...effects of hi-multiplexing within in a cell, while potentially allowing for up to 20-plex experiments with...ever like to write about a recent plasmid deposit please sign up here. Here's what you'll find in this...knock-ins Visual barcodes for live cell clonal multiplexing Improving flexibility for in vivo proximity...Viviana Gradinaru Lab has previously developed the multiplexed version of the Cre-mediated AAV Targeted Evolution...mice and nonhuman primates (for AAV.CAP-B10, for example).  Given that the liver is an immunologically active...

... into Sample6 as graduate students in Dr. Jim Collin’s Lab in 2009. From 2009 to today, Sample6 has changed...leadership, and grown to 25 members.  What is Sample6? Sample6 is an enrichment-free bacterial pathogen detection...their idea at various levels of complexity and abstraction and convince people with diverse backgrounds how...startup company. However, like many like-minded people, I quickly realize that a company requires more...current VP of Business Development & Operations of Sample6, for advice on how to take an idea developed during...etc. What project in the Collins lab inspired Sample6? The initial work focused on using bacteriophages... the focus of the application was changed, but Sample6 still leverages the core engineering and scientific...

...colleagues had moved on as well, so I knew very few people. I didn’t really fit in anywhere and everyone who...the meeting. I didn’t know anything about these people at the time, but I met some very accomplished scientists...with theirs.  It was at this dinner and the next couple of days of discussion with my dinner partners Denise...would provide a structure and support network for people attending large meetings. A key was to provide ...bin with a mentor who had attended the meeting multiple times before and would help the Singletons plan...help with the program on social media. Many of the people who participated as mentors or promoters were connected...vastly expand their networks as they naturally find people with which they have interests and experiences ...

...help of these people and others, we have updated our kit process substantially and have plenty of ideas for...group of people with distinct skills who fulfill a long-term function. When a select group of people are responsible...with other people, whether you want to acknowledge that or not. I just Googled the phrase “People need social... a leader can be successfully doled out to multiple people: someone to coordinate, someone to decide, ... have a specific purpose and finite goals. For example, when it came time to update how we make and distribute...function. Project team A Project Team is assembled to complete a series of tasks with precisely defined goals... in which case whoa! Mission accomplished! One example of a mission-based team in academia would be your...

...the process of communicating science to laypeople.  Laypeople are simply non-experts in the field of science...would all be defined as laypeople when it comes to molecular biology.  Some lay people have a high amount ...other uses. Many laypeople would use “PCR test” to mean “a test that uses a biological sample to look for ...Table 1: Examples of defining your audience for science communication activities  Many people are concerned...language is aimed at a peer audience.  Note that lay people can and do consume scientific communication (it...use-case (definition) in a specific field.  For example, while “PCR test” has recently transitioned from...the possibilities are endless.    Activity Example audience  Talking about your career as a scientist...