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Showing: 261 - 280 of 756 results

  2. Sequencing Options for CRISPR Genotyping

    Type
    Blog Post
    Published
    Oct. 4, 2016, 2:30 p.m.
    ...translational research may require the rigor of both forms of off-target analysis in order to meet clinical...Biased Genotyping Options Technique Sequence Information Detection Limit Advantages Disadvantages Mismatch...experiment is targeted amplicon sequencing. The information from targeted amplicon sequencing is highly sensitive...link genotype and phenotype. It is also key to perform these validations as translational researchers ... NGS-based methods provide the most complete information profile regarding putative off-target sites including...disease, but to treat patients, as well. For more information and a detailed overview of the aforementioned...the Addgene Blog Learn to Use Cas9 Activators Perform Genome-Wide Screens with CRISPR Generate Mouse ...

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

    Type
    Blog Post
    Published
    March 7, 2017, 3:30 p.m.
    ...The undigested (-, lane 1) DNA duplex and those formed from control (GFP gRNA-targeted) BRAF sites (lanes...what I think are the main tips for each step of performing and analyzing a gene edit using Addgene’s lentiviral...lentiCas9-blast. Anti-Cas9 Western blotting was performed on whole-cell extracts from stable pools of A549...blasticidin selection. Anti-Cas9 Western blotting was performed on whole-cell extracts from the initial stable...should appear less green. Note that there won’t be uniform editing across all the cells in the population,...techniques and how quick and easy it is to share information and regents, it’s exciting to think that no matter...

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

    Type
    Blog Post
    Published
    Oct. 24, 2017, 1:54 p.m.
    ...Department of Astronomy. She studies how planets form around young stars and the instruments we use to...scientist participation in government, evidence to inform policies, a protected place to work, funding for...hyper-competitive environment where getting hired hinges on performing near miracles. When evidence does not affirm ...evidence-based research is vital in combating misinformation and propaganda that limits access to full reproductive...

  6. Advice on Career Paths and the Green Card Process for International Researchers and Entrepreneurs

    Type
    Blog Post
    Published
    Dec. 20, 2017, 2:33 p.m.
    ... EB-1B requires that you provide two out of six forms of documentary evidence: Evidence of receipt of... of this post are intended to convey general information only and not to provide legal advice or opinions...particular circumstance or fact situation.  The information presented may not reflect the most current legal...No action should be taken in reliance on the information contained in this post and we disclaim all liability...intentions directing federal agencies to propose reforms to the H-1B visa system. Large firms and firms ...to most postdocs. Commercial successes in the performing arts- not relevant at all. Please do not include...you are a classically trained ballerina who has performed at Lincoln Center. Your case should only focus...

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

    Type
    Blog Post
    Published
    Sept. 28, 2020, noon
    ... including (1) a CRISPR RNA, (2) four proteins forming the QCascade DNA-targeting module with the crRNA... of bacteria including in pooled, genome-wide, formats. Interested in knowing more? Check out our Mobile... WA (2013) Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression...

  8. 15 Hot Plasmids from 2017

    Type
    Blog Post
    Published
    Jan. 9, 2018, 3:02 p.m.
    ...long fluorescence lifetime, and a stable monomeric form as determined by the organized smooth endoplasmic... 9 amino acid N-terminal variation in the Klf4 isoform (KLF4s or KLF4L). Polycistronic cassettes that ...Polycistronic cassettes that contained the KLF4L isoform had more robust initiation of reprogramming (evaluated...All three mScarlet variants have shown great performance in cellular functional imaging and in protein... These Lmo proteins were identified using a bioinformatics approach examining genomes possessing CRISPR...

  9. Save Time and Money by Making Your Own Competent Cells

    Type
    Blog Post
    Published
    Jan. 28, 2014, 3:10 p.m.
    ...We perform over 100 transformations each week at Addgene, so we need to be cost and time efficient. We... stored at -80oC until we are ready to perform transformations. A typical 200mL bacterial culture produces...can find the subsequent transformation protocols below: OpenWetWare Transforming chemically competent ...Untergasser Lab Transformation of chemically competent cells (protocol)  Transformation of electrocompetent... easily transformed without a heat-shock step. Because of the large number of transformations being performed...that transforms well in other competent cells) to ensure that your cells can be readily transformed. Compare...Want even more tips for transforming your cells? Check out our transformation guide.   ...

  10. Pooled Library Amplifications

    Type
    Blog Post
    Published
    May 30, 2024, 1:30 p.m.
    ...libraries, chemical transformations can also be used. Efficiency for chemical transformations is less than that...for your transformation(s). The amount of competent cells you need total and per transformation may vary... DNA, you will likely need to perform several individual transformations, such as splitting 400 ng of ...the manufacturer’s protocol for transformation and expected transformation efficiency, or more specifically...library First, you’ll need to collect specific information about your library: the size of your library ...with a large backbone might be more difficult to transform.    Figure 1: Factors to consider when ... coverage, since this will help decide what transformation efficiency you need to achieve. Most pooled...

  11. Anatomy of a Plasmid Page at Addgene

    Type
    Blog Post
    Published
    Feb. 4, 2016, 3:30 p.m.
    ...research! Sequence information: Depositors may provide full or partial sequence information for their plasmids...successful cloning. Cloning information: This section provides useful information about how the vector was... NEB Stable cells. Resource information: This section contains information about the lineage of the plasmid... bamboozled by all of the different kinds of information on our plasmid pages? Well, to help make the ...through them and make the best use of all the information provided by your colleagues. Top of the plasmid...based off of the plasmid sequence and feature information given by our depositors. Many depositors also... pages, but these are found in the "Resource Information" section of the plasmid page (discussed below...

  12. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

    Type
    Blog Post
    Published
    Aug. 30, 2022, 1:15 p.m.
    ... but it’s really just formaldehyde. Formalin: You may also come across formalin, which refers to a saturated...cause some confusion. Paraformaldehyde: Many IF protocols call for paraformaldehyde (PFA) as the fixative...saturated solution of formaldehyde (saturated being 37%). Formalin solutions often contain methanol as ...order to slow the polymerization of formaldehyde.  Note: Formaldehyde in solution will naturally polymerize... maintaining cellular and tissue morphology. Formaldehyde is the most commonly used cross-linker for IF...fixative. PFA is a polymer of formaldehyde that, on its own, is not a fixative. However, when PFA is dissolved...solution and heated, it depolymerizes, producing formaldehyde. Often people will call the resulting solution...

  13. A Needle in a Base-Stack: Cas9 Structural Biology

    Type
    Blog Post
    Published
    June 4, 2024, 1:15 p.m.
    ...bridge helix, purple.   Apo Cas9 changes conformation frequently (Osuka et al., 2018), but it’s most...electron microscopy and FRET, can offer more information on these regions.   Cas9’s transition from weak...lobes move toward each other into the “closed” conformation shown in Figure 3. Though Cas9 is not a static...binding state, RNA loading stabilizes the closed conformation.   Fun Fact! Even though the Cas9:RNA complex...complex is well described in the closed conformation, recent electron microscopy studies have also found Cas9... Cas9:RNA complexes in the open conformation (Cofsky et al., 2022), indicating that there is more to learn...protein can switch between the closed and open conformations to bend the DNA, destabilizing the base pairs...

  14. Summer SciComm Series: Modes of Communication

    Type
    Blog Post
    Published
    July 1, 2022, 1:15 p.m.
    ...distributed in digital or analog media formats. Cognitively demanding format. Equipment, expense and expertise...with is "how?" How are you going convey your information to your audience? There's so many ways options...incredibly varied in how they prefer to consume information. Personally, I would much rather read the instructions... science.                    Two of the platforms we use at Addgene: YouTube, for longer educational...octogenarians is going to be a hard sell!) Digital platforms often have demographic data of their users readily...geographic location. For example, not all social media platforms are available or used in all countries; not all...mitosis.   Consider also the amount and type of information you want to convey. Small tidbits and fast/fun...

  15. CasPEDIA: A Functional Classification of Cas Enzymes

    Type
    Blog Post
    Published
    Jan. 25, 2024, 2:15 p.m.
    ...provides summary information on Class 2 Cas enzymes. The information is organized in wiki format and includes...display information for a given Cas (SpyCas9a) from CasPEDIA.   Helpful resources and information For ...could arise where you still need more information. If you are performing a viral delivery of Cas, the size...the features? CasPEDIA has collected relevant information on all the Cas enzymes you know and love, and... heard of. Each entry displays the following information for a Cas enzyme: Nuclease properties (CasID...properties of Cas enzymes. That’s a lot of helpful information! To get you started, we’ll give a quick overview... substrate (nick, bind, cleave, mixed). This information represents the ‘big picture’ features of the ...

  16. March for Science

    Type
    Blog Post
    Published
    April 21, 2017, 2:30 p.m.
    ...More concretely, scientists gather information about reality informing people and their governments on what...article are those of the author do not represent a formal stance taken by Addgene or its staff. In Washington...reality and science is how we learn about reality. Formally, a government can be defined as a social contract...into in order to protect their rights (2). Less formally, governments are the organizations that make and...other citizens. Thus civilization proceeds in an informed fashion. For science to be useful to governments... of the bargain and do the research required to inform the public (8–13). Facing the possibility that ...march include: General inclusion of science to inform policy (17). The facts surrounding a matter or ...

  17. RUBY-Red Siliques

    Type
    Blog Post
    Published
    March 1, 2022, 2:15 p.m.
    ...Arabidopsis seed following floral dip transformation next to an untransformed seed from the same plant beginning...Selectable transgene markers for plant transformation fall into two main categories: antibiotic/herbicide...resistance genes usually involves killing the untransformed seedlings or callus (undifferentiated plant ...For example, with CRISPR-Cas9 gene editing, transformation may be identified by resistance to an antibiotic...antibiotic. Successful editing of transformants then typically occurs in T0 plants (from callus) or T1 plants...contaminate it. I have used it for selecting transformation events and am currently planning to use it ...beginning to germinate. (Top right, second panel) Transformed seedlings can be selected without antibiotics...

  18. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    Published
    Oct. 18, 2018, 12:37 p.m.
    ...procedure to be performed using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As...false positive colonies. Transformation - the linear fragments are transformed into E. coli and plasmid...important is the transformation efficiency of the strain: when asking the cell to perform multiple recombination...assembly of a circular plasmid in vitro, before transforming it into E. coli for propagation. However, while...adopted by any laboratory. All procedures are performed using the same three step protocol:  Fragment... encode homology arms to allow immediate co-transformation with linearized vector backbone. DpnI digestion...competent cells of 107 CFU/μg are perfectly fine, yet performing multiple modifications will require ultracompetent...

  19. Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene

    Type
    Blog Post
    Published
    April 6, 2021, 12:30 p.m.
    ...anyone who has ever worked to sequence DNA on NGS platforms will tell you, while the process has become much...sequencing. This process is completed in a plate format yielding anywhere from two to six plates of 96 ...donated MiSeq.  For Complete Plasmid Sequencing we perform a 2x251 run on our MiSeq that takes about two days...involves a significant amount of QC. First, we perform Picogreen quantification on each and every isolated...samples for Viral Genome Sequencing (VGS). QC is performed throughout the library preparation process and...Aligning the sequence The first step we take when performing quality control of newly deposited plasmids is...detecting common features using Snapgene. If we find information that differs from what we would expect given ...

  20. Lab Automation at Addgene

    Type
    Blog Post
    Published
    March 22, 2018, 1:28 p.m.
    ...bacterial transformation One protocol that has been automated is high throughput transformation. With our...a couple of platforms that fit 48 well q- trays allowing us to do up to 96 transformations in one run ...easier and more efficient. Here, I’ll discuss transformation, DNA purification, and filling vials with agar...deposits (over 100 deposits per month!), manual transformations were taking a lot of time. Enter the Biomek... We wanted a machine that would improve our transformation protocol and, after looking at a few options...run compared to doing about 50 transformations in the same amount of time by hand! Setting up a basic ... fine piece of machinery we have been able to transform 96 samples in about 4 hours with a greater than...
Showing: 261 - 280 of 756 results