...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,...

...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...

...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...

... 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...

...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...

...Resources on the Addgene Blog Learn More about Homology Directed Repair Learn More about Non-Homologous End...editing team at the Allen Institute for Cell Science. Learn more by visiting the Allen Cell Explorer at allencell.org...useful coding sequence (i.e. GFP) into a gene while leaving other features of the gene intact. Here, we show...based on known functional aspects of the protein gleaned from the literature and engagement with researchers... Institute for Cell Science Plasmid Collection, please visit the Cell Catalog on the Allen Cell Explorer...hiPS cell lines generated using these plasmids, please visit the Cell Catalog on the Allen Cell Explorer... study cellular structures and processes and can lead to the better understanding of cell biology. We ...

...Finish Your Thesis” online course.  To learn more about her site please visit www.FinishYourThesis.com.   ...all of the above categories. In addition, I also learned about two other kinds of difficult supervisors:... their minds. When you come to a meeting with a clear agenda, you will immediately get your supervisor...supervisor to support you. 3) Explain the problem and leave your emotions out of the discussion Do not assume...a high likelihood of miscommunication, which can lead to even more conflicts. If your supervisor is a ...clam), you will probably need support from your colleagues or other supervisors to complete your work. Whether...package your challenges with your supervisor into learning opportunities as you are advancing career, you...

...MOIs. The presence of the 428 bp Surveyor nuclease cleavage product indicates a gene edit. GFP gRNA-transduced...basis of the Surveyor assay is that Surveyor nuclease cleaves double stranded DNA (dsDNA) at basepair mismatches...mismatches at the site of the edit. Using Surveyor nuclease followed by gel electrophoresis, these mismatches...mismatches are indicated by the presence of DNA cleavage products. The appearance of the 428 bp band indicates...I designed (listed above) worked well and I got clear bands using the conditions above. 2. Design your...product, you would still be able to identify the cleavage product. 3. The Surveyor kit had a lot of good...Before digesting the DNA duplexes with Surveyor nuclease, save some of the duplex and run it as an undigested...

...world's leading yeast researchers in the growing field of synthetic biology and is the UK lead of the ...Benoit to interview Addgene Advisory Board Member and lead researcher at Imperial College London, Dr. Tom Ellis...construct a synthetic yeast chromosome. Listen to learn more about Dr. Ellis, synthetic biology, synthetic...advice for anyone pursuing a career in academia. Learning how to build a lab and a research program and ... thanks to our guest, Tom Ellis! Dr. Ellis is a lead researcher at the Centre for Synthetic Biology and...    Additional Resources from the Addgene Blog Learn about the Synthetic Biology Education Nonprofit ...Nonprofit BioBuilder Plasmids 101: Golden Gate Cloning Learn About Working at a SynBio Startup in Our Interview...

... so if you are interested in learning more about any one of them, please click on the links throughout...with a special emphasis on these five categories. Please note that many of the following cloning methodologies...ends. These fragments are then combined with an exonuclease, a polymerase, and a DNA ligase to carry out ...FastCloning, first described by Chaokun Li and colleagues. Here you simply PCR-amplify your vector and ...When mixed, topoisomerase I provides both the endonuclease and ligase function while 5’ adenosine overhangs...additional enzymes required. FastCloning may be the least expensive of all the options discussed as it only..., as one tube of enzyme should be enough for at least 50 cloning reactions. The ligase cost is comparable...

...University of Zurich), especially with my deep learning-wizard colleague Ahmed Allam. Continued training led to...desired edit, it simply becomes impossible (or at least unreasonable) to test them all. This is where PRIDICT...efficiency and building prediction tools with machine learning (PRIDICT, PRIDICT2.0).   How to apply PRIDICT...prediction tool (ePRIDICT). In summary, machine learning-based prime editing prediction models (such as...biology. His expertise includes applying machine learning techniques to make sense of biology, with a special...SpCas9 activity prediction by DeepSpCas9, a deep learning–based model with high generalization performance... editing efficiency and product purity by deep learning. Nature Biotechnology, 41, 1151–1159. https://...

...the IRES element, scientists have adapted "self-cleaving" 2A peptides into their muliticistronic vectors...mulitple genes from the same mRNA. The term "self-cleaving" is not entirely accurate, as these peptides are...peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence...sequence and the next peptide downstream.4 The "cleavage" occurs between the Glycine and Proline residues found... common 2A peptides employed by scientists. 2A cleavage is universal in eukaryotic cells, and, although...although some scientists report close to 100% cleavage with some of these peptides, no consensus has been reached... added to the 5' end of the peptide to improve cleavage efficiency. How do I get started? If you are looking...

...bioinformatics and HTS, we all had much to learn. And, many wanted to learn more, even those not directly involved...continue such discussions. Beyond the excitement of learning about new cutting edge science, researchers of...in journal clubs. They also have likely had the pleasure of attending both productive and not so productive... (HTS) data we generate and maintain. It became clear that while several people across teams had various...same characteristics as a successful meeting: a clear purpose and effective time management, for starters...feedback you receive include the sense that people learned something new? Did the meeting begin and end on... published science. They also serve as a way to learn about work outside of one's own field in a structured...

...based on the experiments of Kim and colleagues using zinc finger nucleases to harness non-homologous end joining... (DSB) approach at a variety of loci. We were pleasantly surprised by the efficiency of the method. One...polymorphism analysis, allele-specific PCR, T7EN1 cleavage assay, sequencing, RT-qPCR, or immunoblotting ...genetic configurations. Watch the following video to learn Daniel and Matthew's procedures. The video details...genetics of blood cell development and disease. Learn more at his webpage, bauerlab.org.     Matthew Canver...chromosomal deletions in human cells using zinc finger nucleases. Genome Res. 2010 Jan;20(1):81-89. PubMed. 2. ... interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J.Biol.Chem. 2014 Aug...

...with other proteins or nucleic acids. Read on to learn more about this versatile technique.  Immunoprecipitation... only to the Fc portion of the immunoglobulin, leaving the antigen binding sites free to capture the target...conformations and interactions. For difficult to release proteins, including those in the nucleus, more ... an IP, as proteins in an IP are not denatured (learn more about antibodies here!) When using Protein ... immobilize the antibody, it is recommended to preclear the sample to remove any proteins that may interact...nonspecifically with the capture antibody or Protein A/G. To preclear, incubate the sample as you would in an IP using...interactions from residual non-specific proteins but leaves the target protein/capture antibody intact. The...

...applications. Tag cleavage must be performed after primary purification and requires a second clean up step to...protease cleavage at sites immediately following the tag. Unlike MBP, the GST tag can be cleaved off from...need for an extra clean up step of the tag – a handy feature. If you decide not to cleave the GST tag after...Waugh, 2002). The tag can also be proteolytically cleaved after purification, so as not to perturb protein...to remove the cleaved tag from solution. MBP antibodies are available for detection and/or purification...

...CRISPR Featured Topic Page Learn about Non-Homologous End Joining Learn about Base Editing Read 3...can be caused by intracellular factors such as nucleases and reactive oxygen species, or external forces...: The 5’ DNA end of the break is resected by nucleases to create a ssDNA 3’ overhang. This will serve... a primer for DNA repair synthesis, ultimately leading to the formation of double Holliday junctions (...second DSB end. The individual HJs are resolved via cleavage in one of two ways - each junction resolution ...single HJ forms during this process and resolves by cleavage of the crossed strand. While this pathway may ...point to note is that Cas enzymes can continue to cleave DNA if the gRNA/PAM site remains intact. In other...

...ideas in the easiest way possible? Are the shots clear enough for the audience? As Addgene’s Science Video...SynBio, it was time to start our Design-Build-Test-Learn cycles. If you’re not familiar with this, it’s a...process!   Figure 1: The Design-Build-Test-Learn cycle adapted for video making.   Cycle One:... pulled it off! Check out the PCR Primer video! Learn: Producing videos like this would prove to be difficult...phones. Test: I loved the end result, but it was clear this wasn’t going to work for our other videos. ...1280x720 resolution at best. Some came in even lower.  Learn: While this works well for social media videos, ...chat with me remotely via an audio chat (Fig. 4).  Learn: This works great for on-camera presentations and...

...different promoters followed by leader sequences (encoding small leader peptides) downstream of which you... been used to date . If you have any questions, please write to us in the comments section.  FX cloning...gene of interest. Translation of the standardized leader peptide prevents secondary structures in the mRNA...Shine-Delgarno sequence found at the 3’ end of the leader sequence. Using a variety of GFP and RFP fusions...Katarzyna, et al. "Small changes in enzyme function can lead to surprisingly large fitness effects during adaptive...reported in the literature include RNA detection, RNA cleavage, and translational repression or activation: ...Choudhury, Rajarshi, et al. "Engineering RNA endonucleases with customized sequence specificities." Nature...

...applications. We recently released a new variant of jGCaMP7 (keep reading) and will be releasing more variants in...viral vectors. Here are some of the AAV we have released so far in 2019. You can also browse our entire...pAAV-mDlx-NLS-mRuby2 CAG-NLS-GFP pAAV-hSyn-mCherry Read on to learn more!  Calcium sensor AAV The GFP-based calcium...inhibitors that respond to blue light. We recently released a constitutive (pAAV-Syn-Archon1-KGC-GFP-ER2) ...coming soon! These vectors listed below should be released at Addgene in the next 6 months, pending quality... particular catalog item is available for order please email [email protected] with the vector name and...viral vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols...

...This decrease in pH quenches SEP but not DsRed, leading to emission of only red fluorescence. The ratio...ratio of red to green fluorescence allows for clear distinction between vacuolar/lysosomal and cytoplasmic...lysosomes. Therefore, the formation of autophagosomes leads to an increase of yellow puncta (GFF positive/RFP...autophagosome with a lysosome. An increase in autophagy leads to more yellow and red puncta, while a block at ...at fusion of autophagosomes with lysosomes leads to an increase in yellow puncta but a decrease in red ...Addgene! GFP-LC3-RFP-LC3∆G: A fluorescent probe that releases an internal control Another autophagy biosensor... following translation, this fusion protein is cleaved to generate one molecule of GFP-tagged LC3 for ...