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  1. Tips for Using BLAST to Verify Plasmids

    Type
    Blog Post
    ...menu. Typically, we use the default nucleotide database “Nucleotide collection (nr/nt)” as it contains ... the Basic Local Alignment Search Tool (BLAST) provided by NCBI. BLAST helps us compare the sequencing...reference sequences, such as full plasmid sequences provided by the laboratories that deposit their plasmids...programs available, we primarily use Standard Nucleotide BLAST (blastn), Standard Protein BLAST (blastp...BLAST (blastx). NCBI has a terrific getting-started guide for BLAST, which includes a simple explanation of...BLAST search pages. At Addgene, we use blastn to identify any discrepancies in Sanger sequences, such as...ORFs) and determine the potential effect of any nucleotide discrepancies. The blastp and blastx programs...

  2. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    ...A,T or C – but avoid NAG) or guide RNA target (“seed”) sequence. The idea was that these “blocking mutations...mutation), blocking the guide RNA binding site works well. However, since some guide RNAs may tolerate a ...end-joining (NHEJ) pathway, leading to nonspecific nucleotide insertions, dele­tions or other mutations, referred...repair template, such as a sin­gle-stranded oligodeoxynucleotide (ssODN), which contains the intended sequence...efficiency.   But what’s better: PAM-blocking or guide RNA blocking mutations? This depends entirely on...however we have also demonstrated the efficiency of guide RNA blocking mutations. So if the locus does not...while taking advantage of the improved efficiency provided by blocking mutations. Essentially, you:  Step...

  3. How Addgene Creates Content

    Type
    Blog Post
    ...social media, videos, newsletters, guides, and e-books – comes into play. Our goal is to provide informative...using the example of our Antibody Guide, which was published alongside our new antibody services in the... The guide was then assigned to our science communication specialist, Rachel, to write. Identify expertise.... Since our guides are heavily used, our graphic designer made most of the Antibody Guide figures. This...topics for the Addgene blog? Or why we make certain videos? In this post, we invite you behind the scenes ...you, whether you realize it or not! Many of our ideas come from social media conversations, conferences... all of our frequently asked questions!    Identify needed content Let’s take a look at how we create...

  4. Custom CRISPR Screens & the Green Listed Software

    Type
    Blog Post
    ... particular, one slide of the presentation made a lasting impression on me. The slide showed a complex...approach is to identify an appropriate list of genes to target. Thus, it is always a good idea to keep a bioinformatician...Sweden. It can be very time consuming to design 5 guide RNAs (gRNAs) targeting each of the 1000 genes you...screens targeting a (long or short) list of genes provided by the user. This approach is useful for several...effective by world class CRISPR labs.  Check Out This Video to See How Green Listed Works at a Glance!  CRISPR...subsequently sequenced by next generation sequencing to identify which gRNAs are differentially expressed in the...to dissect a complex signaling pathway, and to identify what part of a pathway to inhibit to block the...

  5. Building Global Connections with the International Mentorship Program USA-EUROPE

    Type
    Blog Post
    ... more fully identify with and be inspired by the mentor’s successful example. For evidence of this, you...Valencia): “IMP showed me a wide range of professional opportunities that are outside of our typical boundaries... Program (IMP) USA-Europe was created. IMP as an idea was conceived and developed by its Director, Dr.... closer than ever (although not physically) the ideas and experience from talented professionals to their...together with the universities, a bright future. IMP provides matched one-on-one international mentorship, with...globalized world where individuals can exchange ideas, network, collaborate, and innovate internationally...offered by programs like IMP USA-Europe not only provide the specific technical and logistic support to ...

  6. Analyzing CRISPR Editing Results with ICE from Synthego

    Type
    Blog Post
    ... that you provided for each sample. Guide Target - This is the user-defined 17-23 nucleotide sequence ...sequencing files and provide basic information such as your sample names and guide sequences, and ICE will...which also includes our CRISPR knockout guide design tool, guide validation tool, and guaranteed editing... Lead at Synthego. Article source: Step-by-Step Guide for Analyzing CRISPR Editing Results with ICE on...editing field by making experimental workflows considerably easier, faster, and more efficient than previous...examined ICE analysis of Sanger sequencing data alongside the analysis of NGS-based amplicon sequencing ...time or as a batch of hundreds, and indicate the guide RNA sequence you used. The ICE tool will calculate...

  7. Story of a SynBio Startup: RevBio's Epiphany (or Lack Thereof)

    Type
    Blog Post
    ...joint. Silly ideas (mining local salt deposits to sell at farmers markets), and serious ideas (how to stop...for the future. From good ideas to a SynBio startup At some point, our ideas got pretty good. We started...journal clubs outside of work. Our scientific training was put to use as we explored ideas, planned out...we could do to make ourselves marketable to the wider world and occasionally thinking about how we could...establishment hiring system altogether. We had a lot of ideas about things we could do for work, and they weren... together we make a pretty good team. We have confidence that we can take on any challenge and quickly...marketable product. During this process, we had an idea that survived the literature review, survived technical...

  8. Evolution of Lab Techniques

    Type
    Blog Post
    ...chain-terminating or dideoxy sequencing, was a major breakthrough for determining the order of nucleotides in a string...Sanger sequencing uses modified nucleotides, called dideoxynucleotides (ddNTPs), lacking the 3’ hydroxyl...added and when. Compiling the identity and location of a given nucleotide, a researcher (or a simple computer...the widespread use of Sanger sequencing. Building upon this technology, radiolabelled nucleotides were...Edward M. Southern (1), can be used to detect DNA identity, size, and abundance. If a scientist wants to ...gene mutation, southern blotting can be used to identify the presence of that mutated gene. In southern...One example of southern blotting being used to identify a gene mutation is the genetic test used for sickle...

  9. Transgenic Organisms, Cas9 Gene Drives, and Appropriate Safeguards

    Type
    Blog Post
    ...whenever applicable?Against this, consider the cost of an accidental release. Science relies on popular... DNA cassette encoding the cas9 gene and single guide RNAs (sgRNAs) with appropriate flanking homology...homology into a germline cell can create an RNA-guided gene drive. In principle, this event could happen even...available. Consequently, the best way to avoid accidentally creating a gene drive is also the easiest: don't...barrier protocols, they can reduce the risk of accidental escape to a negligible level. One major reason...publish the concept and likely capabilities of RNA-guided gene drives in the summer of 2014 (after extensive...hope was that anyone else who came up with the same idea or wanted to build a gene drive in a model organism...

  10. Components of CRISPR/Cas9

    Type
    Blog Post
    ...to the guide RNA, Cas9 cannot cut. 2. Bind to target DNA in the presence of a guide RNA provided that target...the components necessary to: 1. Bind to a guide RNA The guide RNA, which will be described in more detail...of the site targeted by the guide RNA. In the absence of either the guide RNA or a PAM sequence, Cas9 ...synthetic guide RNA or gRNA (sometimes sgRNA) In the native Type II CRISPR/Cas system, Cas9 is guided to its...into one RNA sequence known as the guide RNA (gRNA) or single guide RNA (sgRNA). Throughout the remainder...Cpf1) to cut the genome and a small RNA (gRNA) to guide this nuclease to a user-defined cut site. After ...Please note that while this post is intended to provide a general overview of CRISPR components, new Cas9...

  11. Bricking Science: Portraying Scientific Reality Through LEGO

    Type
    Blog Post
    ...Producing stop motion videos is very time consuming; producing a 1-2 minute video takes an entire week ...with the images thereby making the videos more real. To give you an idea of the amount of work, see how many...’s gotten easier now that I identify with the main character in the video. We’ve grown attached to the...Dalila Cunha de Oliveira. Bricking Science is an idea built, literally, 'brick-by-brick' to introduce ...mysterious source of failure the lab gnome. See this video where he/she/it/something is captured on camera:...most people don’t get to experience this very human side to scientific research. Find Protocols Lab Tips ...was born. We chose the stop motion format for our videos as it is the most approachable for LEGOs. Stop ...

  12. BeHeard Award 2018: Diseases of Glycosylation, Arginine Mutagenesis, & Neural Development

    Type
    Blog Post
    ... development. Paula Videira: Congenital disorders of glycosylation Paula Videira is an Assistant Professor...been working with the Rare Genomics Institute to provide plasmid grants to researchers working on rare diseases...through the BeHEARD Award. Congratulations to Paula Videira, Michael McMurray, and Richard Gronostajski who...steps in the glycan biosynthetic pathway and have a wide range of clinical manifestations. PMM2-CDG is the...) activity due to mutations in the PMM2 gene. Videira’s group recently reviewed and reported that PMM2...studied and understood.  In line with this, Prof. Videira and collaborators will use Addgene plasmids to ...altered in PMM2-CDG. These studies are expected to identify new therapeutic targets/solutions and enable scientists...

  13. Antibodies 101: Validation

    Type
    Blog Post
    ...the bulk of the peptides identified to be from the target protein. This approach is ideal for validating...the sample was identified as your target protein, allowing you to proceed with confidence.  Share your ...experiments that you’ve been planning, it is a good idea to pause and make sure that your antibody has been... appropriately validated so that you can have confidence in your results. But what is appropriately validated... This post will go over some of the general considerations you should review before using an antibody.... that your antibody is not binding to proteins outside of your target protein. Show suitability for an...There are a number of resources available that provide in-depth discussions on strategies for antibody...

  14. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    ...If you have a wide variety of protein amounts on the same blot, you may want to consider using a different...online/ask an expert Review protocol videos for a visual guide to good technique High background ...lightly marked on the membrane (1) using the gel as a guide. The gel is then removed from the membrane (2) and... buffers and strengths. If that’s not working, consider using a subcellular fractionation kit or a lysing...re seeing protein aggregation, you may want to consider changing your incubation temperature. Some proteins...aggregate at 95 °C, a common lysis temperature, so consider a longer incubation (10–20 minutes) at 70 °C, ...If you need very accurate protein measurements, consider using an unstained protein ladder, which will ...

  15. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    ...Cas9 orthologs to identify a more efficient, well tolerated, Cas enzyme. They identified the Cas9 orthologue... essential genes in a wide variety of bacteria including in pooled, genome-wide, formats. Interested in...dCas9 variant as well. Furthermore, they provide a high-fidelity (HF) variant of the BE3 that further improves... Then, pCRISPR is introduced along with an oligonucleotide repair template. Through recombineering, the...in Streptomyces  Streptomyces bacteria produce a wide variety of bioactive natural products. To easily...INTEGRATE (Insertion of Transposable Elements by Guide RNA-Assisted Targeting) and the Zhang lab's system...select genes in E. coli. Furthermore, to demonstrate wider applicability they demonstrated success of Cas9 ...

  16. Cpf1: A New Tool for CRISPR Genome Editing

    Type
    Blog Post
    ...our CRISPR guide for a refresher on CRISPR/Cas9.  Figure 1: Cpf1 is a single RNA-guided endonuclease...crRNA/tracrRNA hybrid guides needed for Cas9 function. Since both Cpf1 and its guide RNAs are smaller than...Cpf1 functions differently. Since Cpf1 loci are widely distributed across bacterial species, Zetsche et... crRNAs for Cpf1-mediated cleavage are 42-44 nucleotides in length, about the same size as Cas9’s, but... cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. With...SpCas9. Since Cpf1 doesn’t require a tracrRNA, crRNA guides are only ~42 nt long. Direct synthesis of these...and off-target efficiency When Cpf1 was first identified, we didn't know much about its on-target and ...

  17. With an Eye Towards the Future, We Look Back at the March for Science

    Type
    Blog Post
    ...a march in support of science, I loved the idea and decided to go. At my local March for Science in Albany...advocate for scientist participation in government, evidence to inform policies, a protected place to work,...hired hinges on performing near miracles. When evidence does not affirm our perception of how things are...scientists are tempted to seek out alternative evidence and discard inconvenient findings. Yet, we cannot...scientists to rally in Ottawa to protest the “death of evidence,” and to speak out in unprecedented force during... and actions beyond marches, as well as allies outside the scientific community. Vanessa Sung is currently...Centre at McGill University in Montreal, Canada.  Outside the lab, she dabbles in science policy, public ...

  18. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    ...DNA edits and to perform functional screens to identify genes involved in a phenotype of interest. This...discuss differences between these approaches, and provide updates on how best to design gRNAs. You can also...gRNAs! Comic by Maya Kostman.   Important considerations before you start an experiment with CRISPR ...expression. Location and sequence are important considerations for designing your gRNAs. For indels, it's ...off targets. For CRISPRa and CRISPRi, these considerations are of roughly equal importance (target should...since potential target sites occur ~1 in every 8 nucleotides, restricting gRNAs to 5 – 65% of the protein ...with a PAM preference of NGG, is still the most widely-used Cas enzyme, the development of SaCas9, NmeCas9...

  19. QC Sequencing Technologies at Addgene

    Type
    Blog Post
    ...changes. Base-calling software can then identify the nucleotide that passed through based on these characteristic...used for either guided or new de novo assembly. If a full anticipated sequence is provided, it’s used as...our QC process if there is a 100% nucleotide match to base identity and plasmid length. Any mismatch or...Previously, we provided a general overview of the QC process at Addgene. All plasmids go through this...developed in 1977 by Fred Sanger, was the first widely used DNA sequencing technology. The process relies... bases (A, C, T, or G) is supplied as a dideoxyribonucleoside triphosphate (ddNTP) so that after its addition... reads a fluorescent label on the terminating nucleotide and records it in a “trace” file to determine...

  20. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    ...usually pick 3–6 guides around the region to find the most efficient guide (most guides would work, but...and the concentration of Cas9 and guide RNA (gRNA). These considerations only matter if the PAM sequence...HR template vector design guidelines? A7: A few notes below are considerations for designing HR donor. ...plasmid, clone in your target guides, test run them, and then select the best guide to co-transfect in HR donor...100 bp away, ideally less than 10 bp away. For small (<50 bp) changes, you should consider using ssDNA...for your CRISPR genome engineering experiments, consider that wildtype Cas9 with optimized chimeric gRNA... drive expression, is it necessary to add a G nucleotide to the start of my target sequence? A3: The human...
Showing: 261 - 280 of 1104 results