... the principles of gating, you’ll first need to learn a little bit of theoretical background on the different...might have to do some testing or ask experienced colleagues.   Figure 1: The laser pulse illuminates...prior to running my flow analysis, I can however clearly identify my lymphocyte population (10.7% of all...to generate signals in a reasonable range. If colleagues in your group already have experience with using...my negative, unstained population, which I can clearly differentiate from the positive one (Figure 8).... that in many cases a stain will not result in clearly distinct positive and negative populations. Oftentimes... majority of my cells of interest lies within a clear negative population. When you’re interested in measuring...

...First, it uses a dedicated 5’ exonuclease instead of using the exonuclease feature of T4 DNA polymerase...restriction sites (Type IIS restriction endonucleases) to cleave DNA outside of the recognition sequence..., Jin S, Reiser M, Lockey RF. CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless... was first described by Dr. Daniel Gibson and colleagues at the J. Craig Venter Institute in 2009. Here... which contains three different enzymes: an exonuclease, which chews back the 5’ ends of the fragment...over 200 nucleotides. This may be because the exonuclease could chew through an entire fragment shorter...Cas9 enzyme along with a specific gRNA was used to cleave the 22kb vector. When followed by the standard ...

...lookout for friends or colleagues that have the training or skills you want to learn. Mentors can be senior... “Learning is not attained by chance, it must be sought for with ardor and attended to with diligence...once you have found someone you admire and want to learn from. Check out Joanne's Reddit AMA Who should ...or coffee with someone I don’t know very well at least twice a week. I have done this with people I work...Harvard Medical School researcher Connie Cepko to learn about her mentoring style. Mentors at home and away...or see your work product in action) Can help you learn the local “ropes” and become an insider faster ...this Catalyst Report “Making Mentoring Work” to learn about what makes a good mentoring program. If your...

... had always been a student and I truly enjoyed learning.” Addgene’s Director of Product Management Eric...thing I wanted more of [after undergrad] was to learn and speak the language of science. I didn’t come... me, “it made more sense to get my master’s and leave. I was really devastated when that happened. I didn't...Instagram profiles here (Kenkel, Rinaldi)! But leaving a PhD program doesn’t mean you’ve failed. “I got... see where my life will take me. I’m also just learning. I have a huge interest in data science,” which...the lab, she told me, “I was really interested to learn more when I was introduced to bioinformatics in ...open and collaborative.” She knew she wanted to leave academia, and once she joined an investment club...

...back in 2007, which function as centres for peer learning and knowledge sharing, in the form of workshops...maintains a list of active spaces near you.   Learning about C. elegans with the OpenWorm Project One...variety of aspects from the molecular biology to learning how to grow and care for the worms. As an example...relies upon biologists and computer scientists learning and communicating with one another.      Currently...optimizing the health of domesticated cats and learning about feline microbiomes. One of the aspects of...the kittybiome project, but the primary goal is learning in this case.   Funding Makerspaces One of the...controller and made it spin again!  We’ve also learned to be resourceful and to repurpose old equipment...

...strengthen relationships with your colleagues and supervisors and lead to you being remembered as a person... fill this gap by highlighting that researchers learn many transferable skills while working in the lab...most straightforward, it can mean that everyone leaves disappointed (we’ve all won an argument but felt...sometimes there’s no agreement at all and everyone leaves unhappy.   Negotiation as a creative problem-solving...underlying reasons for this person’s request? Your colleague is if someone can feed their cells this weekend...maintaining and growing relationships with your colleagues within and outside of your organization. In my...still being able to take my vacation? I want to learn more about a different area of the company. How ...

...capable of cleavage, they tested the clones against an NNN PAM library in bacteria where cleavage leads to the...Addgene Blog Learn about Engineered Cas9 PAM Variants Read about CRISPR Base Editing Learn about Homology...increased editing specificity. We’re excited to learn more about xCas9 - here’s what we know so far! ...GAT, GAA, and CAA PAMs, they saw that xCas9 3.7 cleaves multiple PAMs at a higher efficiency than SpCas9...xCas9 are similarly flexible. Like the xCas9 3.7 nuclease, xCas9 3.7 fused to third generation cytidine ...modifications that can be made to Cas9. It is a pleasant surprise that selecting for one beneficial trait...Guide Find CRISPR Plasmids for Mammalian Systems Learn about CRISPR History ...

...working in the laminar airflow are also among the leading causes of contamination. These aerosols generated...naked eyes and hence cannot be detected until it leads to contamination. Another source of the problem ...the cells should be in continuous culture for at least two weeks to give time for low levels of contaminations... the medium should not have been changed for at least two or three days before sampling (Uphoff and Drexler...result interpretation. Also, the signals from the nuclear area make the detection of mycoplasmas difficult...realize it. Here are some things to do: Wear a clean lab coat and mask while handling cell lines. Avoid... cross-contamination. CO2 incubators should be cleaned regularly and dampness of any kind should be avoided...

...reversibility of the genome editing process. Read on to learn about the various light-controlled CRISPR tools ... Sato lab unveiled a photoactivatable system to cleave a target DNA sequence (Nihongaki, et al., 2015)...system is unique in that it utilizes a “split” nuclease - the authors fragmented Cas9 into N-terminal ... moieties move apart, allowing DNA binding and cleavage. This technique was applied to create photoswitchable...inhibited by the unfolding of LOV, allowing the release of Cas9. A noteworthy application of the CASANOVA...in U2OS cells. Upon light activation, dCas9 is released within minutes and red foci formed 20-40 min later...PCK upon reaching the amber stop codon, TAG. (To learn more about site-specific incorporation of amino ...

...and concentration of released DA in mouse brain slices Visualization of DA release dynamics during locomotion... hormone,” it’s also involved in reinforcement learning, decision making, and motor control.  What do...Though DA firing patterns (or the frequency of its release) are well understood, the direct effect of DA binding..., enabling detection of individual peaks of DA release.  Using dLight1 sensors in experiments The Tian... mice Report modulation of DA signaling during learning and reward consumption in live mice Find dLight...following types of data: Report endogenous DA release from a single dopaminergic fiber in mouse brain...stimulus in vivo in transgenic zebrafish Report DA release in live mice over the course of Pavlovian conditioning...

...works with Addgene in some way. For users, we just released a major redesign of how search works on our site...and professional backgrounds. Sometimes this can lead to wildly different ideas of how to approach a problem...information. It is quite possible that the clarifications lead to the story being scrapped or broken up into smaller...After the code is written, it is reviewed by at least one other developer. This is another point of collaboration...different parts of the code. I think the best way to learn about how any code works is to poke it and see what...places, we might be calling it an aliquot. This can lead to confusion as to what the code is supposed to ...on a common name. While we do our best to write clean, reusable code when working on a feature, sometimes...

...genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases." Nature biotechnology 33.2 (2015)... control for these faults. However, it’s still unclear if similar pitfalls exist for CRISPR screens and...off-target cutting of only a handful of guides. It’s unclear if off-target cuts confound the results of high-throughput...without a reduction of on-target activity, but it’s unclear if this holds true in genome-wide screens.   ... are used as controls. Less significant p-values lead to more false-negatives results (i.e. failing to...to identify a gene that’s a hit); but they also lead to fewer false-positives results (i.e. mistakenly...non-targeting guides as controls. While it’s not clear how safe-targeting guides will affect phenotype ...

...Your task is to convince them to accept you as a colleague and give you a million dollar startup package....experiments or a laundry list of molecules to screen has unclear utility. You cannot predict what will happen. Without...validity of a single model. The first approach can lead to new directions, while the second approach could... terminate with the rejection of a model and no clear alternatives. Typically, a research vision is broken...knowledge gap. Aims should have short titles, a clear easy-to-understand question/goal, an approach, anticipated...looks like. If you are successful, what will be learned and/or now be possible to do? Research accomplishments..., anticipated outcome, and payoff for each aim. Learn to communicate your story with only the most salient...

...just those in the focal volume. This leads to more rapid photobleaching and a reduction in signal intensity... bottom with excitation light which can lead to photobleaching when acquiring multiple images over a large...2-Photon microscopes restrict excitation (and bleaching) to a single focal point. This is accomplished...the sample is excited at one time, limiting photobleaching. Similar to widefield, the entire field of ...Fixed tissue sections (> 15 um), 3D cultures, and cleared tissues These samples always require some form ...photon imaging, which restricts excitation and photobleaching to the focal plane can be used to overcome ...overcome this (8). The recent development of tissue clearing techniques (9) now allows researchers to image tissues...

...fluorescent proteins were best imaged after tissue clearing with SeeDB2 (Ke et al., 2016, 2018).   How to...to the excessive amount of XFPs expressed. Then, clear the samples and visualize. For more details on this... the amount of tTA. Paradoxically, too much tTA leads to a reduced expression level of XFPs, most likely...amount of tTA to achieve highest expression levels. Clearing large tissues - Fluorescent proteins are very ...with ScaleCUBIC1 (Susaki et al., 2014), before clearing with SeeDB2. For more information, visit our ... of neuronal circuitry with an index-optimized clearing agent." Cell reports14.11 (2016): 2718-2732. PubMed...26972009. Ke, Meng-Tsen, and Takeshi Imai. "Optical clearing and index matching of tissue samples for High-...

...PhD student at MIT. While I had always enjoyed learning about biology in high school, it wasn’t until ...seemingly abstract biological reactions that I learned about from textbooks just made everything click...all, students build circuits in physics class to learn about electronics or mix chemicals together in chemistry... be freeze-dried into pellets (FD-CF pellets), leaving them shelf-stable until water is added and protein... make the connection between the facts they’ve learned in the textbooks with real world results that they...educational activities using the BioBits platform, please get in touch with us! We’ll work with you to get...experiences and knowledge with each other to help make learning biology more fun – bit by bit. Ally Huang is ...

...CO2 released from dry ice, and whether this affected the pH of our viral samples. Read on to learn how...headspace when the samples were frozen. This was clear by the gradual change in pH visible at the sample...tested whether venting the tubes prior to thaw released the CO2 from the headspace, thus mitigating the...freezer after storage on dry ice allowed the CO2 to leave the tubes, and would prevent the pH change upon ...that tubes subjected to a recovery period of at least 96 hours had the same pH as control tubes that were...possible, we recommend both venting the tube for at least 30 seconds prior to thaw, and also using the thawed...R, Van Vliet K, Chipman P, Agbandje-McKenna M, Cleaver BD, Clément N, Byrne BJ, Zolotukhin S. A simplified...

...beginning to learn about biochemistry. The least this app will do is allow you to write your colleagues’ names...application that allows you to share your data with colleagues and link your notebook from your smartphone to...Gibco Cell Culture by Gibco Whenever I start to learn a new technique, I try to look for a resource to...mass spectrometry app. I digress. When I started learning proper cell culture technique, I tried to find...restriction enzyme or share it via email with a colleague. The app is also great if you’re adding a restriction...researchers in the lab. If you have a favorite lab app, please share it in the comments section below! Many thanks....     Additional Resources on the Addgene Blog Learn How to Keep a Lab Notebook for Bioinformatic Analyses...

...the four strands of the cleaved duplex, while the fourth strand (the released strand in Figure 1 below...Corn’s lab. CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts genomic DNA. The interplay...discovered that the catalytically active form of the nuclease dissociates very slowly after introducing a cut...experiments with more detailed investigations into the release of DNA post-cut. Using substrate DNA labeled on...in vitro. Figure 1: Cas9 binding to the cleaved DNA duplex. Image from Richardson et al., 2016....single stranded donor DNA: Complementary to the released strand, 127bp in length, With 36bp on the PAM-...disastrous (i.e. if breaking the gene is worse than leaving the mutation alone). CRISPR delivery by nucleofection...

...p-hacking, or unclear methods. Many thanks to our guest blogger David Mellor! David Mellor leads the policy...reasons that research can be difficult to replicate. Unclear methods Until someone attempts to replicate the...questionable research practices (QRPs) can quickly lead us to find statistically significant results that...and the new one, previously unaccounted for, may lead to different results. Different setting, different...important.  If we eliminate the problems above: unclear methods, QRPs, and small samples, then when replications... do we make research more reproducible? Combat unclear methods with open data, materials, and code Only...specific statistical test.  Preregistration makes clear the distinction between hypothesis testing work,...