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We narrowed to 929 results for: Comp;

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  1. CrispyCrunch: High-throughput Design and Analysis of CRISPR+HDR Experiments

    Type
    Blog Post
    ... a Cambrian-like explosion of software tools accompanying the CRISPR revolution. (See omictools for even... a 96-well plate and comes out of it. Biotech companies are known to have tools that do this internally...guides and primers in batch, optimized for HDR. Compared to wealth of resources for guide design, there...batch analysis because of its ease-of-use and comprehensive reports. With CrispyCrunch, you get the all ... ranking formula that does just this: Rank = (compressed CFD score) * (gaussian normalized cut-to-insert...is within the coding region, the algorithm then compares the guide sequence to every possible 23bp sequence...Francisco. Before starting his career in software, he completed a MSc in Psychology at McMaster University in ...

  2. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    ...derived from AAV9, their efficacy is generally compared to the AAV9 serotype or an engineered precursor...doesn’t lead to enhanced transgene expression, compared to AAV9, in BALB/cJ mice but is more effective..."Mouse Strain-Specific Tropism?" column. For a complete list of mice strains with LY6A-dependent tropism... by the Gradinaru lab (Deverman et al., 2016). Compared to AAV9, it effectively transduces neurons and...toward astrocytes, but its neuron transduction encompasses more molecularly-defined neuronal subtypes and...astrocytes and neurons, with greatly increased efficacy compared to AAV9 (Hanlon et al., 2019). This capsid was...brain regions, with reduced off-target expression compared to AAV9 in infant rhesus macaques. The AAV.PAL...

  3. New Optimized Genome-wide CRISPRko, CRISPRi, and CRISPRa Libraries

    Type
    Blog Post
    ... stranded DNA breaks that commonly result in a complete loss of function while nuclease-deactivated Cas9...have several limitations. CRISPRko results in a complete loss of function meaning that if an essential ... more hits than the SAM library approach in a comparison study screening for vemurafenib resistance in...(Konermann et al., 2015). Root and Doench also compared the Calabrese library to screens performed with...that ORF and CRISPRa libraries can be used in complement to study gene function. Find the Calabrese pooled...gene function while using fewer sgRNAs. These new compact libraries will allow scientists to conduct genome-wide...transcriptional activation by an engineered CRISPR-Cas9 complex." Nature517.7536 (2015): 583. PubMed PMID: 25494202...

  4. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

    Type
    Blog Post
    ...mutations in the genome, but they’re inefficient compared to CRISPR/Cas9 edits that rely on double strand...only occur through editing by a APOBEC-Cas9n-UGI complex. So how exactly does this work? Figure 1:...editing efficiency can be easily determined by comparing cells expressing only eGFP expression to cells...DNA editing efficiency of APOBEC-Cas9 editisome complexes in real-time (St. Martin et al., 2019). To create...DNA editing efficiency of APOBEC-Cas9 editisome complexes. The eGFP reporters rely on the correction of ...frequency of genomic base pair edits of interest compared to cells segregated solely by a reporter of transfection... single base editing by APOBEC-Cas9 editosome complexes. Scientific Reports 9: . https://doi.org/10.1038...

  5. Deciphering the Mysteries of Behavior with Viral Vectors

    Type
    Blog Post
    ...neuronal function, with far fewer side effects compared to lesioning methods -  they may be the key to...camera or tracking software. Locomotion is a highly complex process on the level of individual neurons and ...different motivation (and brain areas associated) compared to an animal running to escape a threat. Locomotion...plus maze, the light-dark box is a simple device comprised of a brightly lit (risky) and a dark (protected... control group preferred staying in the dark compartment (Howerton at al. 2014). Addgene has several recombinase-expressing... passive mode, the stronger is the depressive component of the animal behavior. With the availability ...behavioral neuroscience to reveal insights into the complex relationship between genes, neurons, and behavior...

  6. Hot Plasmids: Fall 2024

    Type
    Blog Post
    ...Construction of solid matrix-antibody-antigen complexes containing simian immunodeficiency virus p27 using...structures of CRBNmidi alone (A) and in a ternary complex with the multiple myeloma drug mezigdomide and ...on its own, with drug ligands, and in ternary complexes with other proteins, establishing CRBNmidi as ...matured considerably faster, with expression levels comparable to GFP during early embryogenesis (Figure 3).... through the middle of each embryo are shown, comparing GFP, wrmScarlet (mScarlet), and the new mScarlet3...Merritt D.M., Pe K., Cesar M., Hobert O. (2024) Comparative analysis of new mScarlet-based red fluorescent...than FRET-based mechanosensors (~60 kDa) and are compatible with common microscopy methods. Plus, the luminescence...

  7. Q&A With Julie Cicalese, the Newest Member of the Addgene Board of Directors

    Type
    Blog Post
    ... newly formed compensation committee. The compensation committee creates a compensation philosophy that...employee compensation and also serves as an objective body to oversee some of Addgene’s compensation decisions... environment.   What do you hope Addgene will accomplish in the near future? Right now it’s a matter of...

  8. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

    Type
    Blog Post
    ...the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences, Cas12a activity ... amplified region of interest. Cas13 uses a complementary crRNA sequence to bind to the sample and upon...steps and the Cas protein used. Here, all the components needed for the reaction are mixed in a single...incubated at one temperature. Two Cas12a-crRNA complexes are designed, each binding to one strand of the... RPA primers, the Cas12a-crRNA binds to the complementary region in the target sequence and this activity... substrates for Cas13. Cas13, upon finding complementarity between the gRNA and a given fluorescently ...streptavidin coated beads. Fluorescently labelled dCas9 complexes containing sgRNA that have no mismatches bind ...

  9. Antibodies 101: Introduction to Immunofluorescence

    Type
    Blog Post
    ...websites compiling antibody data that can help you compare antibodies from different companies, such as... a fluorophore (Figure 1).   Figure 1: Comparison between how direct immunofluorescence and indirect....  How do you find validated antibodies? Many companies that produce and distribute antibodies validate...through their publications or as a review on a company website. It is important to review the data and... a couple of antibodies that seem promising to compare in your own lab. Even with all of the resources...

  10. Replacing Paper: Tips for Choosing an Electronic lab Notebook

    Type
    Blog Post
    ...lengths to keep accurate records, but given the complexities of modern research, it’s often not enough. A... have software tools that help us efficiently accomplish our daily goals. These tools remind us of our...others using excel sheets or organizing folders in complex ways, there are solutions that bring more to the...-based solutions or open source solutions. In comparison to paper-based record keeping, ELNs offer functionalities... if you are working for a large institution or company, site licenses might be available as well. In most...information. Does your lab need to meet any specific compliance requirements – e.g. 21 CFR part 11, HIPAA? Answering...of time and effort and give the lab a serious competitive advantage.  Many thanks to our guest blogger...

  11. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    ...are designed to be complementary, but each primer may extend beyond the complementary region as long as...products (green) are digested with enzyme “A” as compared to the products of parental plasmid digestion ... Primer Design As a rule of thumb, 11 bp of complementary sequence on either side of the desired mutation...template should be isolated from a methylation competent bacterial strain (e.g. DH5α which is dam+). Methylation...A-overhangs (a feature used in TA-cloning). Such non-complementary bases at the 3’-end interfere with plasmid reconstitution...that any residual parental plasmid (usually from incomplete DpnI digest) can also form colonies under these...

  12. Using Phosphoserine to Study Protein Phosphorylation

    Type
    Blog Post
    ...5). Optimization of this system defined three components required for translation (Figure 1): A tRNA ...the pSer-incorporated polypeptide. These three components were shown to effectively incorporate phosphoserine... the phosphoserine OTS, Sep-OTS, contains the components required for the translation of pSer-incorporated...host strain for pSer-incorporation The second component necessary for phosphoserine incorporation is a...enzyme function, protein interaction, etc. by comparison to non-phosphorylated or ‘wild type’ control ...substitution. Alternatively, you can express the completely wild type protein from the same plasmid.  Upon...dephosphorylation reaction can be tracked through the comparison of phospho:non-phospho species on PhosTag gels...

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

    Type
    Blog Post
    ...transform into competent cells. The master mix of enzymes can be purchased from a company (e.g. NEB or ...so the entire reaction takes an hour or less to complete at 50 °C. After an hour or so, the sample is immediately...master mix for 1 hour, and then transform into competent cells. The success rate of this reaction is typically...terminator sequence), since this would directly compete with the required single-stranded annealing and...ensures correct assembly order of the fragments as compared to the smaller overlapping sequences left with...assembly meets CRISPR Gibson can be adapted to more complicated cloning schemes, such as those where the vector...

  14. IDP and your PI: A Roadmap for Career Planning and Personal Development

    Type
    Blog Post
    ...credit for what we have accomplished. Starting with a recap of accomplishments, from learning new techniques...tangible research and career goals, and celebrate accomplishments. These techniques are laid out in Vincent et...conversation. Goals and Planning Worksheet: Lists accomplishments, research/professional goals and feedback. ...including major project goals and effort needed to complete them. These documents dictate the subject matter...concrete plan in hand, trainees are motivated to complete their work rather than overwhelmed or confused...

  15. PiggyBac-ing Through the Genome Editing Field

    Type
    Blog Post
    ...1]. Like other transposons, piggyBac™ has two components, a transposon and a transposase. The piggyBac...also enables the excision of the transposon in a completely seamless manner, leaving no sequences or mutations...enhanced random integration efficiencies that are accomplished with transfections and are therefore virus-free...enhance gene editing? Gene editing can now be accomplished in a more cost-effective and efficient manner...challenges still remain, such as making gene edits completely seamless (i.e., so no other genetic alterations...transposase (PBx), which contains mutations making it competent for excision, but defective for integration [4...   How does the piggyBac™ gene editing system compare with other methods of gene editing? There are primarily...

  16. Bringing Sustainable Practices to the Lab: Recycling

    Type
    Blog Post
    ...services don’t offer plastic recycling, specialized companies can recycle plastic-- you just have to find them...product packaging) for free! It has to be from their company and it has to be packaging (no coated tissue culture... label. Recycle nitrile gloves Terracycle is a company that takes items not commonly accepted at recycling...your lab or department and send them back to the companies for reuse. They’re really easy to spot and even...recycling program at your institute! Universities and companies have the potential to make a huge impact directly...! These days, it’s important for individuals, companies, and universities to be sustainability leaders...

  17. FastCloning: A Newer, Simpler Cloning Technique

    Type
    Blog Post
    ...methylation competent E. coli for this procedure to work properly. Directly transform chemically competent cells...tips for applying it in your own lab. Table 1: Comparison Between Standard Restriction Cloning and Fast...are then transformed directly into chemically competent E. coli and transformants selected with the appropriate...insert with an additional 15-17 bases that are complementary to the overlapping vector ends. See an overview...an hour at 37°C. This should be enough time to completely digest the template DNA. Note—templates must ...

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

    Type
    Blog Post
    ... of powdered media. Filtration does not ensure complete sterilization as mycoplasma can even escape 0.2... growth of mycoplasma but cannot eradicate it completely. When mycoplasmas are seen in the culture, it...mycoplasmas are used. A broad range of primers encompasses almost all the infecting mycoplasma. The primers...assay kits are available from different biotech companies which are enzyme-based mycoplasma detection kits...synthesized by eukaryotic cells. First, the kit components rupture the mycoplasma and then specific enzymes... of mycoplasma. These methods cannot guarantee complete removal of mycoplasma but, can get rid of most...Microbiol .44: 42–49  Uphoff Cc, Drexler Hg (2002) Comparative Antibiotic Eradication of Mycoplasma Infections...

  19. Finding Your Science Policy Path

    Type
    Blog Post
    ...throughout your career. Plus, just one role can encompass many of these activities leading to a rich day-to-day...combat sexual harassment in academia or a large company who decides how they are going pursue regulatory...science policy roles exist. Explore these resources compiled by the AAAS Science and Technology Policy Fellowship...working in science policy doesn’t have to be a complete career pivot. There are many ways to pursue your...system? Defining your why is what will make you a competitive candidate for any science policy position and...expertise and then once selected, you can pivot to a completely new field without issue. This may be more challenging...much smoother.  Action Items:  Explore this comprehensive resource developed by Dr. Kelly Singel which...

  20. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    ...sequence. This means cloning occurs in vivo, as compared to restriction enzyme cloning where the genetic...homology to the target site for recombination. Key components of the Lambda Red recombineering system The lambda... lambda red recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three...dsDNA was short enough, a ssDNA whose entire complementary strand was degraded. Beta: Beta protects the...created by Exo and promotes its annealing to a complementary ssDNA target in the cell.  Only Beta expression...an ssDNA oligo substrate.   Figure 1: Components of the Lambda Red Recombineering system. See ...region of interest and design an oligo that is complementary to the lagging strand. In practice, it’s easier...

  21. Multiplexed Overexpression of Regulatory Factors (MORF) collection

    Type
    Blog Post
    ... TF library (aka the MORF Collection) and an accompanying expression atlas, including over 3,500 human...from these sites. The Zhang lab generated a comprehensive library of each unique TF open reading frame...endoderm, and mesoderm). These profiles were then compared to that of reference cell types generated by the... the human fetal expression atlas. Computational analysis was used to infer differentiation trajectories...Previously engineered TF libraries were not as comprehensive as this one, so it is an invaluable tool to ...expression dysregulation event. Diseases are often complex, with multiple layers of dysregulation from either...

  22. Management for Scientists: Delegating is Key

    Type
    Blog Post
    ...that no component of the overall project gets left undone or is duplicated. With each component of the ...the project completed by an individual who is a local expert in that task (or has the bandwidth to become...result is high quality and the entire process is completed more efficiently. Listen to our podcast interview...desired outcomes. Concern yourself with what is accomplished, rather than detailing how the work should be...in more engaged, successful employees who can accomplish great things.   To read more on this topic and...

  23. Antibodies 101: Buffers, Storage, and Conjugates

    Type
    Blog Post
    ...and reduce needed compensation.    Working with antibodies can get a bit complicated, but hopefully this...time-sensitive, using precious samples, or being compared to other data in a manner that demands high accuracy... reaction to understand if any of your buffer components could affect the reaction. For instance, sodium...detected in the same assay. This is one of the most complex subjects in antibody-based protocols - but thankfully...another channel's detection range. Though you can compensate for this when reading your data, you’ll want ...

  24. Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection

    Type
    Blog Post
    ...all bacteria are naturally competent to take up DNA, they can be made competent through chemical manipulation...Addgene’s blog to learn about making your own competent cells and our protocols page to learn about bacterial... The phagemids are packaged into replication-incompetent phage particles with assistance from a ‘helper... A., and Karen Guillemin. "Multiple phases of competence occur during the Helicobacter pylori growth cycle...Meibom, Karin L., et al. "Chitin induces natural competence in Vibrio cholerae." Science 310.5755 (2005):...

  25. A Practical Approach to Choosing the B(right)est Fluorescent Protein

    Type
    Blog Post
    ...fluorescent proteins, how to do a head-to-head comparison of fluorescent proteins and how to make a well-informed...your selection on a small-scale head-to-head comparative study under conditions that best mimic the intended...protein properties and provides a quick way to compare fluorescent proteins, e.g. see Chudakov et al (...is of no use. Therefore, it is more sensible to compare fluorescent proteins based on their practical brightness...promising fluorescent proteins and to determine and compare their practical brightness in a system closely ...application. In figure 2 we provide an example of the comparison of the practical brightness of two orange fluorescent...

  26. Luminescent Imaging with Nano-lanterns

    Type
    Blog Post
    ...expressed in cells, generates much less signal than comparable fluorescent proteins. This weak emission hasn... with increased sensitivity and faster imaging compared to previous publications. They also developed ...they successfully imaged multiple subcellular compartments over several minutes using Nano-lantern fusion... fluorescence, so higher concentrations of the compound can be added to the media. Cellular esterase then...producing a constant supply of substrate. Using this compound to excite the Nano-lanterns, Takai et al. continuously... handy. This approach is especially suited to complement optogenetic tools, where light used for excitation...

  27. How to Keep a Lab Notebook for Bioinformatic Analyses

    Type
    Blog Post
    ...track of computation work comes with new sets of challenges. One challenge with computational analyses... of biologists are finding themselves using computational approaches to analyze large data sets (myself...RNA-seq analysis. I had never interacted with a computer at the command line, and so was really starting...especially since you’ll want to dig in to the computation, but they will serve you well as you perform ...directory: A directory is a file system in the computer and is just another name for a folder. For instance...

  28. Seven Tips for Using LinkedIn as a Scientist

    Type
    Blog Post
    ... and trolls. People don’t like to connect with complete strangers as it borders on my first rule of networking...’m asked a lot about requesting connections to complete strangers for informational interviews. Many have...option is in the “More…” menu. You can also follow companies and organizations to see their news and job postings...interviews.  Pro tip: You can learn a lot from the company LinkedIn page. This can include news, inclusion...It’s also a great way to keep track of past accomplishments. Follow all the rules of good resume writing...writing. Include examples of concrete accomplishments in both the technical and “plus skills” areas. Include...

  29. Troubleshooting Your Plasmid Cloning Experiment

    Type
    Blog Post
    ...and the efficacy of your competent cells (when they get old, they lose competency). If all the above is ... be sure that the buffers and temperature are compatible between the different enzymes. Always check the...source (i.e. primer3) or provided by the major companies selling PCR reagents. Nowadays commercial polymerases...a general rule, try to use an excess of insert compared to the backbone plasmid; a starting point could...plasmid. Many vectors contain sequences that are complementary to standard sequencing primers. If not, design...

  30. Celebrating 15 Years of Scientific Sharing

    Type
    Blog Post
    .... Addgene is now as much a tech company as it is a reagent company. I used to have to worry about this...remarkably so for a 90 person company. We were and remain ahead of most companies in creating a welcoming, ...often focus on the excitement of what we are accomplishing and what more we might do. I am grateful every...innovative thinking, and openness to team work.   Company culture Once in awhile I worry a very little ...

  31. Transferable Skills Guide: Public Speaking

    Type
    Blog Post
    ...presentation, lost in a sea of data, my mind went completely blank. After several seconds of truly panicked...honest, if my paper hadn’t been ridiculously more complex than the one before it, I don’t think I would have... panicked state, only option one would let me accomplish my goal.   As a scientist, public speaking isn't...dumpster noises were just last week!  Nerves It’s completely normal to be anxious, nervous, and/or apprehensive... in its most difficult form. You’re presenting complex data, to a group of peers who are there to provide...conferences, or even just provide quick updates during a company meeting increases visibility of your work, opens...

  32. Choosing the B(right)est Fluorescent Protein: Aggregation Tendency

    Type
    Blog Post
    ...based on size. Whether higher molecular weight complexes are detected will depend on the homodimer affinity...During the experiment, the solution is diluted, complicating the interpretation. Because of this, these methods...fluorescent protein is monomeric without the accompanying data to prove it is worthless. The OSER assay...and biological properties of new fusions can be compared with mEGFP or mTurquoise2, which are established...that you will be using. If you do a head-to-head comparison right, it will be valuable information for the... are engineered can be shared to simplify the comparison by others. Together, we can build a valuable ...

  33. Choosing the B(right)est Fluorescent Protein: Photostability

    Type
    Blog Post
    ...us to the key question: what is the best way to compare the photostability of different fluorescent proteins...measurements for different fluorescent proteins and by comparing how the fluorescence intensity changes over time...photostability of the fluorescent proteins can be directly compared. An example of a photostability measurement of...fluorescent protein can be identified by a head-to-head comparison of several fluorescent proteins of the same spectral.... In figure 1 we provide an example of such a comparison between cyan fluorescent proteins using widefield... power, the photostability of the variants is comparable (lower panel of figure 1). To avoid changes in...

  34. CRISPRainbow and Genome Visualization

    Type
    Blog Post
    ...specific chromosome. This image represents the composite of fluorescent measurements from red, blue, and...modified GFP-dCas9 and the gRNA scaffold to enhance complex assembly, thereby decreasing the amount of background... labeling efficiency was similar to that of a comparable FISH method. Importantly, they did not observe...with the previously established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that...that they could evaluate the degree of chromatin compaction even for this small distance! To the authors’...CRISPRainbow generates 6 unique colors and a white composite. gRNAs with two identical hairpin binding domains...

  35. Plasmids 101: CcdB - The Toxic Key to Efficient Cloning

    Type
    Blog Post
    ...specific components that are key to the cloning reaction. Understanding the specific components is essential...take up your insert. But exactly how does ccdB accomplish this? Let’s start with a brief history of the...system contains ccdB. A successful insertion will completely replace ccdB with the investigator’s insert of...interest.   CcdB resistant E. coli strains complete the system But ccdB alone isn’t the only key to...protein involves poisoning of DNA-topoisomerase II complexes.” J Mol Biol. 1992 Aug 5;226(3):735-45. PubMed...

  36. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

    Type
    Blog Post
    ...was contributed by guest blogger Alan Wong. The complexity of biological systems can hinder our attempts...simple tool that allowed us to rapidly decode the complexity? The CombiGEM-CRISPR technology was developed...of providing an easy-to-use tool to analyze the complex combinatorial genetic networks underlying your ...mapping the combinatorial genetics that underlie complex regulation in biological systems. The CombiGEM ...new technologies to interrogate and understand complex biological systems. He can be contacted at aslw...transcriptional activation by an engineered CRISPR-Cas9 complex." Nature (2014). PubMed PMID: 25494202. PubMed ...

  37. CRISPR Meets Synthetic Biology: A Conversation with MIT’s Christopher Voigt

    Type
    Blog Post
    ... do as genetic circuits become larger and more complex? Voigt: If you look at all the products of biology... on genes at different times. It requires more complex synthetic networks. Addgene: What hurdles did you... perspective and you can layer them for more complicated functions. Those things are easy with CRISPR..... There haven’t been enough genetic parts for complexity. We have characterized almost 600 terminators...of a cell. The objective is to give people the complete toolbox for taking on very large genetic engineering...just using what biology has given us. Wood is a complicated material and we can access it, but we are stuck...

  38. Open Resources and Plasmid Tools For Studying C. elegans

    Type
    Blog Post
    ...science and collaboration. The field already has comprehensive reference pages and curated databases for scientists... (CeNGEN) The CeNGEN goal is to establish a comprehensive gene expression atlas of every C.elegans  neuron...that is creating the “first virtual organism in a computer.” This project has produced various resources ...four-dimensional contrast interference (DIC) microscopy and computer imaging processing. The combination of these methods...methods provides unique opportunities to develop computational methods to better understand animal development...Hammarlund, Marc, et al. "The CeNGEN Project: The Complete Gene Expression Map of an Entire Nervous System...

  39. A Look at Addgene's QC Process

    Type
    Blog Post
    ... to stop functioning properly or be something completely different than what was originally intended. ...tags, promoters, and other critical functional components. Fun fact! Some older plasmids in our collection...each of their plasmids, from descriptions of key components of the plasmids to partial, full, or annotated...Addgene receives deposited plasmids, they undergo complete plasmid sequencing using an in-house Illumina ...Addgene. The assembled plasmid sequence is then compared to the information provided by the depositor. ... sequence is not available, QC scientists will compare the NGS results to the plasmid information, other...

  40. Plasmids 101: The Wide World of Natural Plasmids

    Type
    Blog Post
    ...that allow their host organisms to thrive in competitive environments. The genes encoding these functions...triumph over your neighbors is to eliminate the competition, and Penicillium isn’t the only organism that... are also less aggressive ways to survive in competitive environments — like by living where no one else...to be active only in the presence of the target compound. These systems are a gold mine of parts for synthetic...Shareck et al., 2004). Plasmid categorization is complex Plasmid classification has shifted over the years...necessarily reflect genetic relationships or plasmid incompatibility. Plasmids may also have more than one function...

  41. Viral Vectors 101: Calcium Sensors

    Type
    Blog Post
    ...blood clotting, and as we all know, a structural component of bones and teeth. It is an enzyme co-factor,..., a neuron subtype) and specific subcellular compartments for measurements. All of the sensors discussed...calcium concentrations in different subcellular compartments can vary greatly. Additionally, the calcium ...susceptible to artifacts and pH interference, and comparison between experiments can be more difficult due...GCaMP6 only) or mammalian cell expression, and compatible with constitutive or Cre-dependent expression...Genetically encoded calcium indicators to probe complex brain circuit dynamics in vivo. Neuroscience Research...

  42. Progress Towards a PAM-Free CRISPR

    Type
    Blog Post
    ...significant amount of flexibility compared to a strict single-compatibility sequence like NGG. Nonetheless...NG, a rationally engineered Cas9 with NG PAM compatibility, as the name implies (Nishimasu et. al, 2018...know and love about Cas9, but with broader PAM compatibility. This alternative PAM requirement adds a significant...PAMs and is capable of editing at frequencies comparable to SpCas9. SpRY can also edit NYN PAMs, but with... Ai C, Hu J. In-depth assessment of the PAM compatibility and editing activities of Cas9 variants. Nucleic...

  43. Illuminating Epigenetics with A FRET Based Biosensor

    Type
    Blog Post
    ...remodeling. Chromatin is a complex of DNA and histone proteins that compacts cellular DNA into chromosomes...in the compacted region and results in a loss of gene expression. In this way, genes in compacted regions...and tightly bound DNA is to histones, the more compacted it becomes. This prevents the cellular machinery...have huge impacts in the way we study and treat complex or multifactorial diseases, enabling faster diagnosis...

  44. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    ...information directly into the file. Email the completed spreadsheet to [email protected] along with ...used to knockdown expression of mRNAs with complementarity to the shRNA sequence. Empty backbone A plasmid...plasmid data in the pages described below. You can complete these pages sequentially by entering the appropriate...the links at the top of the page. Figure 11: A completed plasmid page for review before submission. Once... right of each data entry page. Once you have completed the data entry process for all of the plasmids...Figure 13: Click the "Submit Deposit" button to complete the data entry. Clicking the "Submit Deposit" ...third party rights holders. Once this process is complete, you will receive a Deposit Confirmation email...

  45. Transferable Skills: Negotiation

    Type
    Blog Post
    ...while also ensuring that my work gets completed? I want my company to pay for me to attend a conference... How can I negotiate my starting date with the company so they know I’m excited while still being able...want to learn more about a different area of the company. How can I negotiate with my manager to gain experience...conference. How can I negotiate to show the company that investing in me is also beneficial to them? I have an...but I need help from another person/team at the company. How do I negotiate with the person/team for help...

  46. PhD Applications After COVID

    Type
    Blog Post
    ...know that this process only became even more complicated with COVID-19 and standards for hybrid, virtual...without the financial and logistical stress that accompanies traveling for campus visits. This is definitely...where the PhD application cycle becomes much more complex. “How do I reach out to potential mentors?” “Where...very valid questions, and often make an already complicated process even more difficult. I’ve learned that...to be incredibly useful, as it gives a more comprehensive idea of that advisor’s research interests which... for the interview. Having notes close to your computer with things you want to highlight in the interview...

  47. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    ...bacterial strains, like NEB Stable. NEB Stable competent cells lack RecA, a protein that aids in the homologous...helps me figure out if I have enough plasmid to complete my experiment. AAV is usually the limiting reagent...produce more virus because I don’t have enough AAV. Compared to the Addgene AAV Production protocol, I take...concentrate the purified virus. This process can be completed in one long day, or the virus can be stored at...set can be used and samples are quantified by comparing to an AAV standard curve, which is generated from... plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR...

  48. PITChing MMEJ as an Alternative Route for Gene Editing

    Type
    Blog Post
    ...about a month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction...HDR and NHEJ, please see the linked blog posts. Compared to its counterparts NHEJ and HDR, MMEJ doesn’t...strategy looks very similar to HDR, but it is accomplished using much smaller regions of homology, which...strategy looks very similar to HDR, but it is completed using much shorter sequence homologies. Abbreviated...efficiency of PITCh-based genomic insertion would compare to CRISPR sticky-end insertion using the nuclease...Genome-Editing Based Approach for Mapping Multiprotein Complexes in Human Cells.” Cell Rep. 13: 621-633. PMID: ...

  49. The CRISPR Software Matchmaker: A New Tool for Choosing the Best CRISPR Software for Your Needs

    Type
    Blog Post
    ...These insights are hard to appreciate without computational support. Indeed, rational sgRNA design is not...experimental applications in CRISPR engineering. When compared to that collective utility, it’s easy to fault...tool unique. The CRISPR Software Matchmaker is composed of these features and enables you to select the... based on their comfort level with operating a computer.  Examples: “offline”, “online”, “CLI”, and “GUI...few offline tools available mostly work on any computer but generally require working knowledge of the... power of each tool they are interested in by comparing their past results to the designs suggested by...pattern at any edit distance. It won’t, however, compute any score other than the edit distance. Added functionality...

  50. Negative Can Be Positive: Open AAV Data with Addgene

    Type
    Blog Post
    ...Sharing data and community concerns AAV vectors are complex biological reagents; many factors, like storage...literature, scientists can use the Data Hub to compare different methods and vectors, and use this data...through word of mouth or old lab notebooks and computer files. If you asked your team members or colleagues...much unpublished AAV data do you have sitting in computers and notebooks right now?, I’m willing to bet the... rat CNS: AAV PHP.EB vector dose–response and comparison to AAV PHP.B. Gene Therapy, 25(5), 392–400. https..., C., & Flotte, T. R. (2015). Stability and compatibility of recombinant adeno-associated virus under ...
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