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We narrowed to 839 results for: LEA

Showing: 81 - 120 of 839 results

  1. DAP arrays for multiplex gene editing

    Type
    Blog Post
    Published
    July 7, 2022, 1:15 p.m.
    ...Although CRISPR-Cas12a, with abilities to process and release its own gRNAs from a single array, has been a top...prime editing, both and their derived tools are leading the edge of the precision gene editing. CRISPR-...and has previously been used for multiplex Cas9-nuclease editing. Typically, a single array encoding the...array. After expression, the individual gRNAs are released by endogenous tRNA processing machineries, specifically...tRNAs Luckily, a fun feature of tRNA was fully unleashed by the Gao Lab’s work. Led by PhD student Qichen...promoter, which is followed by tRNA processing to release individual gRNAs for gene editing. They named this...human or plant source), by tuning the length of 5’ leader sequences of a given tRNA to achieve the highest...

  2. Management for Scientists: Delegating is Key

    Type
    Blog Post
    Published
    Feb. 20, 2015, 8:30 p.m.
    ...the manager to succeed. More people in leadership roles leads to more ideas, initiative and creative ... Are the desired goals and outcomes clear? If you don’t have clear outcomes in mind, you won’t be able... Series Learn How Volunteering at a Professional Organization Can Help You Develop Leadership Skils Set...Harvard Medical School researcher Connie Cepko to learn about her management style. Recipes for delegation...assignee and this is a recipe for bad outcomes. Clearly articulate the desired outcomes. Begin with the...delegate so the person doing the task will have to learn and stretch a little, but be careful not to go too...calendar even if it is just 15 minutes a week. Clearly identify constraints and boundaries. Where are ...

  3. Antibodies 101: Introduction to Gating in Flow Cytometry

    Type
    Blog Post
    Published
    April 4, 2024, 1:15 p.m.
    ... 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...

  4. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    Published
    March 1, 2016, 3:30 p.m.
    ...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 ...

  5. Will You Be My Mentor? Finding and Asking for Mentoring Support

    Type
    Blog Post
    Published
    Feb. 4, 2014, 3:50 p.m.
    ...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...

  6. Meet an Addgenie: Insights into Grad School, Career Paths, and More

    Type
    Blog Post
    Published
    June 25, 2020, 1:15 p.m.
    ... 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...

  7. Experimenting in an Open Source lab: from CRISPR to Cats

    Type
    Blog Post
    Published
    July 23, 2015, 2:30 p.m.
    ...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...

  8. xCas9: Engineering a CRISPR Variant with PAM Flexibility

    Type
    Blog Post
    Published
    March 28, 2018, 6:52 p.m.
    ...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 ...

  9. Transferable Skills: Negotiation

    Type
    Blog Post
    Published
    March 3, 2022, 2 p.m.
    ...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 ...

  10. Mycoplasma Contamination: Where Does It Come From and How to Prevent It

    Type
    Blog Post
    Published
    Oct. 8, 2020, 1:15 p.m.
    ...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...

  11. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    Published
    Sept. 3, 2020, 12:15 p.m.
    ...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 ...

  12. Which Fluorescence Microscopy Techniques is Best for Me?

    Type
    Blog Post
    Published
    Oct. 10, 2017, 1:57 p.m.
    ...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...

  13. Designing Your Chalk Talk for the Academic Job Interview

    Type
    Blog Post
    Published
    March 12, 2019, 1:37 p.m.
    ...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...

  14. An Inside Look into Addgene's Dev Team

    Type
    Blog Post
    Published
    Jan. 23, 2020, 2:45 p.m.
    ...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...

  15. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

    Type
    Blog Post
    Published
    Sept. 13, 2018, 1:55 p.m.
    ...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 ...

  16. Sensing Neuronal Dopamine

    Type
    Blog Post
    Published
    July 30, 2019, 12:59 p.m.
    ...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...

  17. Tetbow: Bright Multicolor Labeling for Neuronal Tracing

    Type
    Blog Post
    Published
    Jan. 24, 2019, 2:24 p.m.
    ...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-...

  18. Creating accessible biology activities in schools with BioBits

    Type
    Blog Post
    Published
    Aug. 2, 2018, 12:56 p.m.
    ...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 ...

  19. How Dry Ice Affects Viral Vector Sample pH & How to Avoid It

    Type
    Blog Post
    Published
    June 20, 2017, 2:30 p.m.
    ...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...

  20. 5 Great Apps for Lab Life

    Type
    Blog Post
    Published
    Sept. 22, 2016, 2:30 p.m.
    ...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...

  21. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

    Type
    Blog Post
    Published
    March 24, 2016, 2:30 p.m.
    ...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...

  22. The Many Reasons for Irreproducible Research- and a Vaccine to Eradicate It

    Type
    Blog Post
    Published
    Jan. 30, 2020, 2:15 p.m.
    ...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,...

  23. CRISPR 101: Off-Target Effects

    Type
    Blog Post
    Published
    Feb. 8, 2024, 2:15 p.m.
    ...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...

  24. Generating Mouse Models Using CRISPR/Cas9

    Type
    Blog Post
    Published
    July 12, 2016, 2:30 p.m.
    ...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...

  25. Supporting Rare Disease Research with the 2020 BeHEARD Awards

    Type
    Blog Post
    Published
    Sept. 8, 2020, 2 p.m.
    ...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...

  26. Transferable Skills Guide: Cross-team Communication

    Type
    Blog Post
    Published
    Nov. 21, 2019, 1:53 p.m.
    ...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...

  27. Transferable Skills Guide: Public Speaking

    Type
    Blog Post
    Published
    April 20, 2023, 4 p.m.
    ... 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...

  28. Giving gRNAs a Facelift - Synthetic and Beyond

    Type
    Blog Post
    Published
    Oct. 20, 2022, 1:15 p.m.
    ...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...

  29. CRISPR Cheat Sheet

    Type
    Blog Post
    Published
    Jan. 27, 2021, 2 p.m.
    ... 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...

  30. Plasmids for Endogenous Gene Tagging in Human Cells

    Type
    Blog Post
    Published
    April 6, 2017, 1:02 p.m.
    ...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 ...

  31. 7 Simple Strategies to Resolve Conflicts with Difficult Supervisors

    Type
    Blog Post
    Published
    Jan. 21, 2015, 1:30 p.m.
    ...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...

  32. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    Published
    March 7, 2017, 3:30 p.m.
    ...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...

  33. Synthetic Biology, Artificial Chromosomes, iGEM & More - Interview with Tom Ellis

    Type
    Blog Post
    Published
    Nov. 29, 2017, 2 p.m.
    ...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...

  34. Plasmids 101: 5 factors to help you choose the right cloning method

    Type
    Blog Post
    Published
    Aug. 21, 2018, 12:31 p.m.
    ... 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...

  35. PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs

    Type
    Blog Post
    Published
    June 25, 2024, 1:15 p.m.
    ...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://...

  36. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    Published
    Sept. 9, 2014, 8:20 p.m.
    ...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...

  37. A Guide to Starting Your Own Journal Club

    Type
    Blog Post
    Published
    Feb. 6, 2020, 3 p.m.
    ...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...

  38. CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

    Type
    Blog Post
    Published
    Feb. 18, 2015, 3:09 p.m.
    ...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...

  39. Antibodies 101: Immunoprecipitation

    Type
    Blog Post
    Published
    Dec. 7, 2021, 2:15 p.m.
    ...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...

  40. Antibodies 101: Affinity Tags

    Type
    Blog Post
    Published
    Sept. 19, 2023, 1 p.m.
    ...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...
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