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  1. Clear and Accessible Writing

    Type
    Blog Post
    Published
    Feb. 15, 2024, 2:15 p.m.
    ...is would change over the next twenty years. But learn we did! From Addgene's very beginning, we had to...information about images that a sighted reader would glean from the image itself.  As we incorporated alt text...to alt text. We also were aware of the need for clear, accessible writing in scientific writing, and how...development workshop, “Make a Greater Impact Using Clear and Accessible Scientific Writing.” In this post... of writing: alt text (pun intended.)  Steps to clear writing Let’s use Figure 1 as our example. Remember...student using viral vectors for the first time and learning how to purify AAV.  Second, you’ll identify your...free to share your alt text in the comments! We learned a lot from listening to others' alt text descriptions...
  2. Working with Nuclear Receptors

    Type
    Blog Post
    Published
    July 15, 2014, 4:03 p.m.
    ...MEDLINE-indexed journal Nuclear Receptor Signaling. Dr. McKenna edits the website of the Nuclear Receptor Signaling... elicit their actions on other tissues. At the Nuclear Receptor Signaling Atlas (NURSA), one of our primary...regulated at the cellular, tissue and organ level by nuclear receptors (NRs), their coregulators, and their ...their physiological endocrine ligands. Nuclear receptors and their ligands NRs represent the largest family...Consortium represents a unique funding model in nuclear receptor research, and has generated a set of comprehensive...address many important and fundamental questions in nuclear receptor and coregulator research. Our Q-PCR datasets...for example, document the expression patterns of nuclear receptors across multiple different tissues in ...
  3. How to Lead a Great Meeting

    Type
    Blog Post
    Published
    Feb. 9, 2016, 3:30 p.m.
    ...continuing the project after leaving the meeting. Follow the tips below to learn how you can run a top-notch...direction you’d like to pursue? Undefined scope can lead to long meetings spent on details that do not yet...our all-company meetings short, informative, and pleasant. Email: Sometimes, the right meeting is no meeting...screen using a Google Doc. Action items: Before leaving the meeting, skim back through your notes and pick... tend to get stuck diving into details, ask a colleague to help keep you accountable for moving along....your organization. Ask for feedback from your colleagues. When you attend a well-run meeting, note what...what worked well and ask the meeting leader for some tips. Before long, you will be running collaborative...
  4. Management for Scientists: Managing vs. Leading

    Type
    Blog Post
    Published
    March 31, 2015, 4:08 p.m.
    ... Connie Cepko to learn about her management style. Leaders look to the future Leaders must get new things... they lead. Effective leaders rarely give orders (although they must occasionally). Good leaders exert...urged you to work on developing your “leadership skills”. Leadership is prized at all levels of an organization...figure out what skills were really leader-specific. How is leadership different from good management? Aren't...Manager, Leader” by Charlotte Beers, divides workers into three cycles: Doers, Managers and Leaders. I like... Mask: How Leaders Spark and Sustain Change, by Peter Fuda and Richard Badham Leadership Insights for...Business Review 10 Must Reads: On Leadership (Articles on Leadership, also can be purchased individually...
  5. Save Time with Transient Plant Leaf Transformations

    Type
    Blog Post
    Published
    July 25, 2019, 12:03 p.m.
    ...wheel. Test different leaf ages Usually younger leaves work better. Not every leaf is equally susceptible...transient leaf transformation, the engineered Agrobacterium strain is brought into contact with the leaf tissue...Agrobacterium infiltration into leaves does not work well, as the leaves have a waxy texture and are difficult...transgenic plant can take even longer, a transient plant leaf transformation process could save the plant biologist...studied in hairy roots, as functional chloroplasts and leaf specific cell types are missing in roots. The development...development of transgenic hairy roots takes at least six months. Generating a transgenic plant takes even...biosynthetic pathway. Why plant scientists do transient leaf transformations The development of transgenic plants...
  6. Learning Technical Skills at a Lab Central Workshop

    Type
    Blog Post
    Published
    Feb. 20, 2018, 2:30 p.m.
    ...opportunities to learn from other biotech companies at workshops and can use what we learn to improve processes... end repair Adapter Ligation Clean up/Size selection Amplification Clean up  I found this kit easy to... post, I’ll tell you a little bit about what I learned from a New England Biolabs (NEB) hosted workshop...CosMCPrep system for this, but thought we could learn a little bit more about high throughput DNA prepping...class at NEB’s new teaching space at Lab Central to learn all about it. LabCentral is a lab and office space...emerging biotech companies. The space was modern with clean bold colors of orange and white. The decor was very... this is the most important part of this newly released prep kit. In their prior NEBNext Ultra II DNA ...
  7. Gaining Leadership Skills Volunteering at a Professional Organization

    Type
    Blog Post
    Published
    Aug. 6, 2015, 2:30 p.m.
    ... led for 5 years.  Different leadership styles Being an effective leader requires you to understand how...would come necessary changes. I also learnt to adapt my leadership style according to the needs of my committee...strengths and work effectively. Leadership responsibilities Being a leader in an organization, you need ...that come with leadership. The NPA was a great organization to develop these leadership skills in particular... These “leadership institute’ weekends would entail intense training to develop our leadership and management...sharing with other more junior leaders what I had experienced and learnt. Opening doors to further opportunities...develop my leadership and advocacy skills but will also put me in closer connection with other leaders in my...
  8. From Lab Bench to Leadership: Transitioning into Management as a PhD

    Type
    Blog Post
    Published
    Jan. 21, 2025, 2:15 p.m.
    ...Scott Courses: Leadership programs on platforms like Coursera, LinkedIn Learning, or edX. Podcasts...for Leaders More resources on the Addgene blog Management for Scientists: Managing vs. Leading Management...to a managerial role. For those contemplating the leap into management, the journey can be as exciting ...intended) expectations —yours and your team's—can lead to frustration. Clinging to technical tasksDelegation... member has unique motivations, strengths, and learning styles. For example, during my time as a lab scientist...required more guidance and structure. I quickly learned that my approach needed to adapt based on individual...individual needs. For instance, I had one colleague who preferred detailed instructions and regular check-ins...
  9. Choosing a CRISPR Nuclease: Site Accessibility, Specificity, and Sensitivity

    Type
    Blog Post
    Published
    Nov. 5, 2019, 1:28 p.m.
    ...Considerations when choosing your Cas nuclease When choosing the Cas9 nuclease for your experiments, there are...but since then, scientists have adapted CRISPR nucleases for many more specific research needs. In this...determined by the PAM sequence recognized by the nuclease. For the most frequently used Cas9, spCas9 from... Figure 1: The PAM sequence that the Cas nuclease recognizes determines site accessibility. Cas9s...off-target activity of Cas9. This can occur when Cas9 cleaves a site that is not a perfect match to the guide...difficult to determine, the potential for off-target cleavage should be an important consideration during any... measure of the on-target activity of the Cas9 nuclease. This can be determined by measuring the indel...
  10. Fluorescent Protein Travel Awards - Protein Variants, a Serotonin Sensor, and an Artificial Leaf Replica System

    Type
    Blog Post
    Published
    June 4, 2019, 1:02 p.m.
    ...neurons (measured using a red calcium sensor) leads to the release of serotonin (measured using the newly developed...shape and metabolism of leaves can be changed by light and temperature and leaf autofluorescence can interfere...explains. Developing an artificial leaf replica means creating a “leaf” with similar permeability of water...for bacteria. To compare the artificial leaf to A. thaliana leaf, Bernach plans to use a suite of fluorescent... determine whether the artificial leaf behaves like the living leave in this aspect. To monitor bacterial... the variant is unstable, it will be degraded, leading to reduced eGFP fluorescence. Cells expressing ...dosages.  Michal Bernach: Developing an artificial leaf replica system to study plant-bacteria and bacteria-bacteria...
  11. Hot Plasmids: Summer 2024

    Type
    Blog Post
    Published
    Aug. 27, 2024, 1:15 p.m.
    ...from AWI by helical LEAs. C-E) cryo-EM single-particle analysis of PRC2 with RvLEAMshort (1:6 molar ratio... imaging. A truncated form of a tardigrade LEA, RvLEAMshort, protected samples and was compatible with...tardigrades!  Find RvLEAMshort here!    Abe, K. M., & Lim, C. J. (2024). Small LEA proteins as an effective...MagIC-Cryo-EM for enrichment and structure determination LEA proteins protect samples during cryo-EM grid freezing...: https://doi.org/10.1101/2024.01.21.576499.    LEA proteins protect fragile samples during cryo-EM grid...lab found that adding Late Embryogenesis Abundant (LEA) proteins, small proteins that naturally adsorb to...under CC-BY-NC-ND 4.0 license. Protection by the LEAs allowed the authors to determine structures at comparable...
  12. A Practical Guide to Optimizing AAV DIO and FLEx Vector Expression

    Type
    Blog Post
    Published
    Nov. 2, 2021, 1:15 p.m.
    ...://doi.org/10.1089/hum.2019.277 Lavin TK, Jin L, Lea NE, Wickersham IR (2020) Monosynaptic Tracing Success...BioRender.com   Causes of recombination and leaky expression While leaky expression and spontaneous recombination...will have some level of recombination that can lead to leaky expression in cells that do not express Cre ...scrambled cre-recombinase sites lead to greater frequency of reversion and leaky expression. On the right non-homologous...recombination between these sites may be the cause of leaky expression. To test their hypothesis, they randomly...recombination. This indicates that the amount of leaky expression can largely be attributed to the level... non-homologous scrambled recombination sights lead to reduced reversion and less off-target expression...
  13. Cpf1: A New Tool for CRISPR Genome Editing

    Type
    Blog Post
    Published
    Oct. 14, 2015, 2:30 p.m.
    ...RuvC-like endonuclease domain, but they lack Cas9’s second HNH endonuclease domain. Cpf1 cleaves DNA in ...orthologs that display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 family members contain...based system, require a single-component nuclease to mediate cleavage rather than the multi-subunit complex...contains a RuvC-like endonuclease domain, but it lacks Cas9’s other HNH endonuclease domain, indicating ...functional CRISPR nuclease that could be adapted for genome editing. The use of a different nuclease could potentially...that display robust cleavage activity similar to that of Cas9. These two nucleases, AsCpf1 and LbCpf1 ...proximity to the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively far ...
  14. CRISPR 101: Targeting RNA with Cas13a (C2c2)

    Type
    Blog Post
    Published
    Sept. 21, 2017, 2:07 p.m.
    ...Cas13a. The origins of Cas13a: An RNA cleaving CRISPR nuclease Cas13a was originally identified in 2015... crRNA to cleave target DNA (Cpf1 requires only a crRNA for target recognition and cleavage). C2c2 (now...domains to cleave target DNA. The HEPN domains within Cas13a are essential for RNA cleavage, consistent...bind and cleave RNA non-specifically. The ability of dCas13a to bind target RNA without cleaving the molecule...between Cas13a and Cas9 is that Cas13a binds and cleaves RNA rather than DNA substrates. In terms of structure... residues in the Cas13a molecule results in a “nuclease dead” Cas13a (dCas13a), that is capable of binding...binding target RNA but lacks the ability to cleave the RNA target.  Table 1: Comparison on Common CRISPR...
  15. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

    Type
    Blog Post
    Published
    May 5, 2020, 1:15 p.m.
    ...gets activated. This unleashes indiscriminate ssDNA cleavage activity which cleaves the supplied fluorescently...fluorophore that is quenched when intact and released when cleaved. Cleavage results in fluorescence and this approach... This activation results in a trans cleavage reaction that cleaves a nearby RNA sequence that contains...activated and performs non-specific collateral cleavage. This cleavage is coupled to fluorescence visualization...fluorescently labelled ssDNA substrates. Upon cleavage, the fluorescence is no longer quenched and can be ... a PAM (Protospacer Adjacent Motif) for target cleavage. This test can be performed in two steps for challenging...ssDNA-FQ reporters that exhibit fluorescence upon cleavage. Upon amplification by the RPA primers, the Cas12a-crRNA...
  16. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    Published
    April 16, 2020, 1 p.m.
    ...) to cleave ssRNA or mRNA. It also exhibits a “collateral effect” of non-specific ssRNA cleavage. How ...ssRNA cleavage byproducts. Thus, binding of Cas13 to the programmed region of interest would lead to both... the Addgene Blog Learn about using toehold switches to detect Zika virus   Learn more about targeting...degradation to develop the method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) (Chen...Both SHERLOCK and DETECTR harness the promiscuous cleavage and degradation of neighboring ssRNA and ssDNA...ssDNA by Cas13 and Cas12a, respectively, to cleave and activate a reporter. The detectable signal from this...to the programmed sequence, it can promiscuously cleave surrounding ssRNA molecules. In SHERLOCK a quenched...
  17. A Needle in a Base-Stack: Cas9 Structural Biology

    Type
    Blog Post
    Published
    June 4, 2024, 1:15 p.m.
    ...for Cas9 nuclease function. Stem loops 2 and 3 are not strictly necessary for in vitro cleavage, but they...the two nuclease domains, Cas9 ensures both are positioned correctly before HNH or RuvC can cleave DNA, ...? It’s not actually clear! In vitro, Cas9 holds tightly to DNA even after cleaving it, so many researchers...profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnology...   Cas9 has two major lobes, called NUC (for nuclease) and REC (for recognition). The NUC lobe contains... and RuvC domains, which are named for similar nuclease folds found in other proteins. The C-terminal ... and helps recognize the guide RNA. Unlike the cleavage domains, the recognition domains—including the...
  18. Cpf1 Update: Comparison to Cas9 and NgAgo

    Type
    Blog Post
    Published
    July 14, 2016, 2:30 p.m.
    ...characterized two Cpf1 nucleases, distant cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered pattern...detectable off-target cleavage. Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites...proximity to the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively far ...Cpf1-mediated cleavage, the continued presence of the PAM may give Cpf1 the ability to cleave again and possibly...the two (tracrRNA and crRNA) needed by Cas9 for cleavage. Now, two new studies show that Cpf1 displays ..., Bernd, et al. "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015)...blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered fashion, creating a 5 nucleotide...
  19. Sequencing Options for CRISPR Genotyping

    Type
    Blog Post
    Published
    Oct. 4, 2016, 2:30 p.m.
    ... strands hybridize, the nuclease can detect strands with mismatches and cleave them. The results are then...surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases." G3: Genes| Genomes...profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity." Nature biotechnology...genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases." Nature biotechnology 33.2 (2015)...genotyping. The most common options include mismatch cleavage assays, such as Surveyor™, T7E1, and Sanger sequencing...adequate standards for validating edits.  Mismatch cleavage assays rely on pairing between the edited strand... in the genome predicted to contain off-target cleavage events. Unbiased techniques search the whole genome...
  20. Cas13d: Small RNA-targeting CRISPR enzymes for transcriptome engineering

    Type
    Blog Post
    Published
    May 3, 2018, 1:48 p.m.
    ...sequence! In bacteria, Cas13d-mediated cleavage promotes collateral cleavage of other RNAs. As with other Cas13s...and two nuclear long non-coding RNAs. One interesting twist to Cas13d enzymes is their cleavage pattern...EsCas13d favors cleavage at uracils, but a more detailed exploration of this cleavage pattern is necessary...a CRISPR repeat array near a putative effector nuclease. The Cas13d proteins they identified have little...identified Cas13a-c orthologs, but they do include HEPN nuclease domains characteristic of the Cas13 superfamily...their own CRISPR arrays into guide RNAs. crRNA cleavage is retained in dCas13d and is thus HEPN-independent...Cas13s, this collateral cleavage does not occur when Cas13d is expressed in a mammalian system. Since Cas13d...
  21. Plasmids 101: Protein tags

    Type
    Blog Post
    Published
    Dec. 11, 2014, 4:26 p.m.
    ...and structural integrity; cleavable. Not great for purification; too cleavable in eukaryotes TAP GRRIPGLINP...FLAG) are cleaved by specific proteases without requiring the addition of an independent cleavage recognition...cell. FLAG tags can be cleaved by enterokinase, which recognizes DDDDK^X, cleaving after the lysine. The...Notes TEV ENLYFQS Cleaves between the Gln and Ser residues Thrombin LVPRGS Cleaves between Arg and Gly...maltose-binding protein (MBP). Overexpression can also lead to insolubility, and aggregated protein is not useful...sequence of amino acids, is basically a molecular leash for your protein. If you’re working with an uncharacterized...-based purification; has inherent enterokinase cleavage site GST Large Protein 26 Purification and Stability...
  22. Components of CRISPR/Cas9

    Type
    Blog Post
    Published
    Feb. 2, 2016, 5 p.m.
    ...have two endonuclease domains: the n-terminal RuvC-like nuclease domain and the HNH-like nuclease domain...conformational change that positions the nuclease domains to cleave opposite strands of the target DNA. Thus...and Cas9, bind to these transcripts and C) cleave them leaving mature crRNAs bound to the Cas9/tracrRNA ...Cas9 complex to the target DNA which is E) cleaved leaving a F) double-strand break. A “gRNA” is a researcher-designed...Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related Cpf1) to cut the genome...genome and a small RNA (gRNA) to guide this nuclease to a user-defined cut site. After reading this post...functions of the various CRISPR/Cas9 components. Please note that while this post is intended to provide...
  23. A History of Genome Engineering in Popular Culture

    Type
    Blog Post
    Published
    Feb. 25, 2020, 2:15 p.m.
    ...serve as slaves.  Meganucleases In 1989 the first ever DNA-editing enzymes, meganucleases, were discovered...created by genetics.    Zinc finger nucleases Zinc finger nucleases (ZFNs) are artificial restriction enzymes...DNA-binding domain to a DNA-cleavage domain (FokI). Zinc finger nucleases were first designed in 2005....activator-like effector nucleases (TALENs) were engineered. TALENs follow a DNA-binding and DNA-cleaving strategy similar...The major drawback of all the endonuclease-based gene editors (meganucleases, ZFNs, TALENs, and CRISPR-Cas...2002) Mutations altering the cleavage specificity of a homing endonuclease. Nucleic Acids Research 30:3870...in 1990, just a year after the discovery of meganucleases. The storyline involves the resurrection of ...
  24. Transferable Skills Guide: Teamwork

    Type
    Blog Post
    Published
    Nov. 28, 2017, 2 p.m.
    ...every team should have a goal, or at the very least, a clear reason for existing. Here are a few types of...own experience: Leadership Few things are more frustrating than being on a leaderless team. Sometimes ...team, I try to make it very clear why that person is there. If it becomes clear after a few meetings that...strong sense of purpose, an excellent leader, and perfectly clear lines of communication, but if teammates...this gap by both highlighting that researchers do learn MANY transferable skills while working in the lab...The famous discoveries and experiments I’d been learning about for years were usually the products of teams...forward nearly 20 years and I am known by some colleagues as the “cross-team guy” at Addgene, an unofficial...
  25. Light Sheet Fluorescence Microscopy

    Type
    Blog Post
    Published
    July 16, 2015, 2:30 p.m.
    ...these authors were able to reconstruct a cleared guinea pig cochlea in 3D. The basic configuration of the...passes through the specimen, which often leads to photobleaching.  Furthermore, point-by-point scanning ... of clear specimens and for in vivo imaging of transparent organisms. Advances in tissue clearing Similar...modified by others to produce Murray’s clear solution.  Murray's clear (11) was mostly used to study the development...with diameters less than 4nm. Lateral view of a cleared rat pons, medulla and the first segments of the...fluorescence microscopy was first pioneered by Voie and colleagues and originally named orthogonal-plane fluorescence...reconstruction (5). In order to image large tissue such as cleared rodent brain, the Ultramicroscope was developed...
  26. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    Published
    Oct. 9, 2018, 12:21 p.m.
    ... follow a lead and when to terminate a project are fundamental skills that all group leaders must master... 8. Learn to mentor, manage, communicate, and teach The dirty secret about becoming a group leader is ...teaching experience and learn about modern pedagogy techniques, especially active learning. Serve as a daily...Assuming you want to become competitive for a Group Leader position, what will you need? Multiple first ...universities and institutes annually advertise for Group Leader positions starting in August, the year before the...completed. Therefore, to be ready to apply for a Group Leader position, key first author manuscripts need to ...perspective for your project. Furthermore, Group Leader position applications require 3 letters of reference...
  27. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    Published
    Jan. 10, 2017, 3:37 p.m.
    ...relies on two distinct Cas9 variants—nuclease-competent or nuclease-null—in order to effect different perturbations...imagine transfecting cells with both the nuclease-positive and nuclease-null variants of Cas9 along with the... Cas9 has two catalytic domains, each of which cleaves one strand of DNA upon binding the protospacer....Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9 interacting with either a full-length...native Cas9's two catalytic domains, HNH and RuvC, cleave DNA strands complementary and non-complementary...the protein’s nucleolytic capability, and this nuclease-null or “dead” Cas9 (dCas9) variant can then be... many cases this is due both to lower relative nuclease efficiencies as well as to more stringent targeting...
  28. Multiplex Genome Editing with CRISPR-Cpf1

    Type
    Blog Post
    Published
    May 9, 2017, 2:12 p.m.
    ...Cpf1 CRISPR Nucleases   Cas9 Cpf1 Components crRNAtracrRNACas9 crRNACpf1 Endonuclease size spCas9... multiple targets compared to Cas9.  Read on to learn more about Cpf1 multiplexing.  For an in-depth review...crNA: ~42 nt Requires tracrRNA? Yes No dsDNA cleavage Blunt end 5' overhang PAM sequence NGG TTTV ...PAM site preserved? Usually Destroyed Yes, Cpf1 cleaves 5' of the protospacer Multiplexing CRISPR-Cas9...express the gRNAs and Cas9. Co-transfections can lead to variable expression levels due to differences...allow for expression of multiple gRNAs (i.e. Csy4 cleavage sequence, tRNAs, multiple individual promoters...requires its own promoter   Kabadi et al Csy4-Cleavable Cassettes transfection gRNAs expressed from a ...
  29. Back to Bacteria: CRISPR gRNA Multiplexing Using tRNAs

    Type
    Blog Post
    Published
    June 2, 2015, 6:06 p.m.
    ...multiplexing gRNAs used the RNA nuclease Csy4 from Pseudomonas aeruginosa to cleave 4 gRNAs separated by 28 base...cell, this system is clearly also robust enough to ensure high levels of gRNA cleavage.   Xie et. al constructed...sequences preceding gRNAs, to test for gRNA cleavage. gRNAs were cleaved precisely as predicted with no addition...schematics. Scissors indicate cleavage sites. After confirming gRNA cleavage, they examined gRNA expression...with Cas9 and the polycistronic transcript. As nucleases may have off-target effects and/or toxicity, it...every living organism. Precursor, or pre-tRNAs are cleaved by RNase P and Z in eukaryotes (E in bacteria),...elements of tRNAs are necessary for recognition and cleavage. This sequence flexibility indicates that gRNAs...
  30. Choosing the B(right)est Fluorescent Protein: Photostability

    Type
    Blog Post
    Published
    June 8, 2017, 2:30 p.m.
    ...a fluorophore will bleach. This is, however, not the full story. The photobleaching rate does not depend... 2-fold does not reduce photobleaching by half. How exactly the photobleaching rate depends on power is...to select a photostable fluorescent protein. Photobleaching is the irreversible destruction of a fluorophore...influence of light. Any fluorescent molecule will photobleach at some point. For live-cell imaging, it is desirable...fluorescent proteins that are photostable. On top of photobleaching, fluorescent proteins may display reversible...destroyed. Factors that affect photostability The photobleaching rate of a fluorophore primarily depends on ...excitation wavelength. A fluorophore is less likely to bleach when it is excited off the peak wavelength, since...
  31. We're Updating Our Hold for Publication Policy

    Type
    Blog Post
    Published
    June 24, 2025, 1:15 p.m.
    ...necessarily a clear sign that you are ready for us to release your plasmids, so we won’t immediately release preprint-associated... to release their plasmids for distribution, and we’ve found that this step was sometimes unclear or missed...situation for all. Addgene will release plasmids on hold if they are clearly described in a journal publication...of risking leaving them on hold indefinitely. Of course, the fastest way for us to release your plasmids...publication. Plasmids will be released 3 years after the deposit date. Don’t worry, we’ll let you know before... before these releases happen (and congratulate you on your paper!). And as always, we won’t distribute...to update the plasmids to add the citation and release the hold. Increasing impact Making the plasmids...
  32. Transferable Skills Guide: Managing a Team

    Type
    Blog Post
    Published
    Jan. 30, 2018, 2:42 p.m.
    ...over time! Communicate often and clearly As a manager you need to clearly state expectations and goals. ...this gap by both highlighting that researchers do learn MANY transferable skills while working in the lab... be patient and keep an open mind. First let’s clear up a misconception about being a manager: yes, being...professional career of. Like everyone else I mostly learned through experience and from my mistakes. Here I... people! More specifically, they had different learning styles. One was a “Doer” and the other was a “... the best protocol;  he wanted to get going and learn through doing things. Had I realized that at the...fits all”. Manage the individuals in your team: learn what motivates them (keep in mind that it can be...
  33. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    Published
    Feb. 27, 2024, 2:15 p.m.
    ... a collection that is greater than 75 plasmids, please let us know by contacting [email protected]. ...plasmid can be added on subsequent pages. Type – please choose from one of the following descriptions for...e-mail us at  [email protected] Special cases Please contact us at [email protected] or +1 (617)-ADDGENE...must be verified by next-generation sequencing. Please see the help center article, "Can I deposit a pooled... any genomic alterations unique to your strain. Please contact us at [email protected] or +1 (617)-ADDGENE...bacterial strain(s) through Addgene. Description – please indicate the gene cloned into your plasmid, any... not lost.   Step 1: Sequences, Maps, and Files Please provide any full or partial plasmid sequences and...
  34. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

    Type
    Blog Post
    Published
    Nov. 1, 2018, 12:35 p.m.
    ... of restriction endonucleases and methyltransferases. Restriction endonucleases cleave DNA at or near ...system is MazE/MazF. The toxin MazF is a ribonuclease that cleaves specific sequences in RNA resulting in...restriction enzyme from cleaving the DNA. A role for restriction endonucleases in plasmid addition was...chromosome. The more stable endonuclease can then cut the chromosome leading to cell death. Antisense RNA-regulated...antitoxin. However, the antitoxin will be degraded leaving the cell to be killed by the more stable toxin....GyrA subunit of DNA gyrase and trapping it in the cleaved complex. This results in DNA breakage and cell ... which binds and inhibits MazF, is continually cleaved by the ClpAP protease so without continual production...
  35. Summer SciComm Series: Cognitive Load

    Type
    Blog Post
    Published
    June 16, 2022, 1:15 p.m.
    ...connect one concept to another which leads us to… Framework Learning to think like an immunologist, a geneticist...that you think are clearly connected, step back and ask yourself, “are these clearly connected because ...the way information or tasks are presented to a learner; and germane cognitive load refers to the work ...which can drive away even the most enthusiastic of learners.  What contributes to cognitive load?  Everything...use up your audience’s mental bandwidth.  Jargon Learning, using, and retaining new vocabulary takes time...others. But sometimes we’re unaware that we are leaning on an unspoken expectation of our audience knowing...details to communicate. It’s okay (and encouraged) to leave blank spaces or to gloss over some aspects when ...
  36. CRISPR 101: Cas9 vs. The Other Cas(s)

    Type
    Blog Post
    Published
    Dec. 8, 2022, 2:15 p.m.
    ...its method of DNA cleavage. Instead of cleaving DNA at a defined site to form a clean DNA break, Cas3 locates...non-specific RNA cleavage and 2). cell toxicity. Cas7-11 does not display collateral RNA cleavage and is significantly...We are all oh so familiar with the nuclease Cas9, but how does it stack up to the new Cas enzymes on ...without shortly thereafter hearing ‘Cas9’. The Cas9 nuclease is a genome engineering powerhouse, and for good...staggered breaks, also known as sticky ends. Cas12a cleavage results in 5 bp overhangs and is particularly ...enzymes, Cas14 targets ssDNA instead of dsDNA. It cleaves ssDNA with high fidelity - sensitive to even a ... to Cas12a is that Cas14 also indiscriminately cleaves non-complementary ssDNA when engaged by its target...
  37. Plasmids 101: Terminators and PolyA signals

    Type
    Blog Post
    Published
    March 31, 2016, 2:30 p.m.
    ...eukaryotic polyadenylation signal directs cleavage at the cleave signal and addition of a poly-A tail to...Resources on the Addgene Blog Learn how the Promoter Controls Gene Expression Learn about New Cloning Techniques...transcription stops, or termination. Read on to learn more! Download our Plasmids 101 eBook to get all... (such as a gene) and initiate the process of releasing the newly synthesized RNA from the transcription...destabilize the transcription complex, initiating cleavage of the transcript.   Figure 1: A predicted...transcription of the template and initiating the desired cleavage event, although some engineered terminators come...and RNA secondary structure to induce transcript cleavage, similar to the Rho-independent termination found...
  38. Is this the right place for me? 8 tactics for choosing a lab

    Type
    Blog Post
    Published
    Oct. 2, 2018, 12:56 p.m.
    ...with them and remaining close colleagues of the PI? Or do lab alumni leave the field or get pushed out ...association? Will you learn how to run a lab if that is your aspiration? Will you help or at least observe grant...Scientists are lifelong learners. I believe we won’t be happy if we aren’t learning so before you take any...very communal. That didn’t suit my collaborative learning style at all. Finally I rotated in a small lab... members stay around? Is it too long or do they leave prematurely? Do lab alumni stay in the same field...organizational skills? Are they involved with colleagues and the department? Are they successful at getting...teamwork that brings out your best performance and learning? Everyone’s different so give some thought to ...
  39. PiggyBac-ing Through the Genome Editing Field

    Type
    Blog Post
    Published
    May 31, 2016, 3:30 p.m.
    ...with the piggyBac™ transposase itself, please read on to learn more about this exciting technology from...site-specific nuclease (such as CRISPR/Cas9) to create a double-stranded DNA break. After DNA cleavage, the piggyBac...the transposon in a completely seamless manner, leaving no sequences or mutations behind. Furthermore, ...before, thanks to the development of site-specific nucleases (CRISPR/Cas9, TALENs, and ZFNs). However, several... the edit of interest. Fortunately, by combing nuclease technology with the piggyBac™ system, these challenges... or cells lines, including stem cells, without leaving behind any accessory DNA sequences [2], [3]. Simply...integration [4], the donor cassette is removed leaving behind no accessory transposon sequences.  The ...
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