...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...

...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 ...

...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...

...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 ...

...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...

...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...

...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,...

...RNA-guided nuclease that either introduces a ssDNA nick, a dsDNA break, or is engineered not to cleave DNA and...confer specificity of the nuclease; however, many Cas and Cas-like nucleases are promiscuous. In other... from base/prime editing or Cas cleavage. In the case of Cas cleavage, off-target mutations can be mitigated...experiment. For CRISPR work, this often means DNA cleavage at unanticipated sites. While off-target effects...these approaches will work for every experiment, at least one will likely be useful. Optimal gRNA selection...swaps will only reduce off targets where nicking/cleaving of DNA occurs, not for deadCas activator/repressor...cause a DSBs will be very low, reducing off-target cleavage. How to quantify off-target events Even after...

...model, and its success is not always guaranteed. To learn more about this process check out our Mouse Modeling...mutation introduced by CRISPR. The sequence started clean and then, about 100 bps into the run, it got “dirty... hard. I was mentally prepared to go through a learning curve that would result in success only after ...about 125 nt that specifies the target, the Cas9 endonuclease that creates the DNA double-strand break (DSB...through non-homologous end joining (NHEJ), this will leave behind a “scar”, with nucleotides missing or added...free” and refrain from talking to friends and colleagues while working with these reagents. Other than...important job of delivering the payload! Last but not least, when using CRISPR, remember to appreciate the fact...

... in front of an audience and convey your ideas clearly and strategically is powerful in almost any career...public speaking, and I'm happy to share what I've learned along the way. Own your time When I was an undergrad... your purpose The purpose of our assignment was clear: present a paper to our classmates so they could...don’t just happen to you or me (though, let's be clear, they have all happened to me.) They happen to everyone...speaking as a scientist is that you will have to learn this skill in its most difficult form. You’re presenting...audience or not in your own native language. And, at least in the biomedical field, with very little text on... speaking as a scientist is that you will have learned this skill in its most difficult form, which makes...

...vulnerable to attack by both cytoplasmic and nuclear nucleases, as they will be recognized as foreign RNA...these woes with photoactivatable and photocleavable gRNAs. Photocleavable guides are generated by introduction...which improves mismatch discrimination and is more nuclease-resistant, resulting in less off-target events...introduction of a single photocleavable 2-nitrobenzyl linker in the 20-bp targeting region of the gRNA. These...These gRNAs can be cleaved and rendered useless after less than a minute of exposure to the appropriate ...brief light exposure (several seconds) the gRNA is released and the CRISPR-Cas9 system can initiate targeted..., Y., Wu, B., et al. Cas9 deactivation with photocleavable gRNAs. Molecular Cell 81, 7, 1553-1565 (2021...

...resulting in the activation of keratinocytes, which release inflammatory mediators that trigger the activation...pharmaceutical interventions that would inhibit IL-36 release,” says Dr. Mark Mellett, a senior researcher at...autoinflammatory loop: keratinocytes both respond to and release IL-36 cytokines. Keratinocytes are the main source...what molecules or proteins are involved in IL-36 release in keratinocytes,” says Mellett. This would help...Bicuspid aortic valve (an aortic valve that has two leaflets, instead of three) and acute aortic dissections...that this is abnormally deregulated in TS and can lead to cardiac developmental defects. Eight genes from... codes for G𝛃1 subunits of G protein complexes, lead to a rare type of developmental delay, and is characterized...

...meetings and hearing the same words, they were leaving these meetings with different interpretations of... remarkably well for this task. Good scientists learn how to communicate their work differently based ... talk about your experiments to your PI, your colleagues, or your non-scientist friends and family is ...context and junking jargon I suspected I knew at least one source of the confusion in our meetings. A lot...questions--which were real, not hypothetical--was clearly “no.” When there was room for misinterpretation...list of commonly used (and misused) words that clearly needed an Addgene-specific definition. Words such...simplified the first weeks or months when I was learning the terminology of a new field. Annotate: When...

... CRISPR nuclease. Note: Most CRISPR plasmids from Addgene produce either a gRNA, a nuclease, or both...Editors: CRISPR nucleases that cut or modify RNA as opposed to DNA. Fusing the nuclease to an adenosine...CRISPR system to cut other DNA or RNA sequences. Nucleases: Proteins that bind to gRNAs and are directed ...sequence. Base Editors: Modified versions of CRISPR nucleases that make single base changes in the DNA sequence...Activate/Repress: Modified versions of CRISPR nucleases that can’t cut DNA (dCas9, for example) that are...gene. Screen: A library of gRNAs with a CRISPR nuclease target multiple genes in a cell population. The...