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We narrowed to 1,010 results for: PLE

Showing: 481 - 500 of 1010 results

  1. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    ...through homologous recombination. The concept is simple: deliver a template with a modified version of ...homology is typical for targeting constructs. In the example shown in Figure 1, large sequences corresponding...is expressed (knocked in). As seen in the above example, you can remove a floxed resistance gene using ...plasmids described in the preceding sections are simple ways of knocking out a non-essential gene of interest...there are 3 loxP sites, recombination can occur in multiple ways. The desired recombination event will remove...tools for creating knockout animals or cell lines, please consider sharing them by depositing with Addgene...

  2. Genome-wide Screening Using CRISPR

    Type
    Blog Post
    ...strengthen your results. Enrichment or depletion of multiple gRNAs targeting the same gene can be strong evidence...jar of jellybeans, a pooled CRISPR library is a complex mixture. Pooled libraries are composed of many ...sorting and screened for a particular phenotype. For example, mutant cells can be used in drug screens to identify.... Loss of representation due to enrichment or depletion of specific gRNAs can lead to skewed results. ...any experiment, the use of appropriate controls, multiple replicates and several cell types can strengthen...

  3. Tips for Using BLAST to Verify Plasmids

    Type
    Blog Post
    ...getting-started guide for BLAST, which includes a simple explanation of the different BLAST programs, databases...consistently run our blastx searches with the “Low complexity regions” filter unchecked so that these regions...benefit is that blastp searches do not filter low complexity regions by default, meaning that you do not have...Clustal is our frequent choice for pairwise or multiple sequence alignments of nucleotide or protein sequences... sequences. We also use COBALT for aligning multiple protein sequences, particularly for comparing different...//www.ebi.ac.uk/Tools/webservices/#multiple_sequence_alignment_msa http://www.ebi.ac.uk/Tools/webservices...

  4. Which Fluorescent Protein Should I Use?

    Type
    Blog Post
    ...allowing very low background level of fluorescence. Examples for such photoactivatable or photoconvertible ... and their emission is green or red light. For example, T-Sapphire, LSSmOrange, and LSSmKate. Fluorescent...sensor), and iGluSnFr (glutamate sensor). More examples of these biosensors can be found at Addgene. ...also is also called Bimolecular Fluorescence Complementation (BiFC). 8 points to keep in mind when choosing...system can excite, and detect the emission. For example, if your microscope has only two lasers, at 488nm...minutes after it is translated to a few hours. For example, superfolder GFP (sfGFP) and mNeonGFP can fold ...

  5. DNA Purification Without a Kit

    Type
    Blog Post
    ... takes 1-1.5 hours to complete, while this syringe-based method can be completed in 20 to 30 minutes. ...drawbacks of kits and typically follow the same principles: cells are lysed, RNAs and proteins are removed... centrifugation. At this point, the plasmid DNA sample can be treated with RNase to remove RNA contamination...waste footprint. The Zhao and Deng labs developed a simple method for regenerating silica columns that come... reduce the number of spin columns you need to complete your research. Do you know of other nucleic acid...

  6. Introducing Addgene's New Search - Find Plasmids More Easily For Your Research!

    Type
    Blog Post
    ...including depositing labs and institutions. For example, search for “Melina Fan” and you’ll be able to ...plasmids from a paper have been deposited? In this example we’re going to be looking for recombinant monoclonal...enough... If in the past you have tried to combine multiple terms in an Addgene plasmid search and found the...each filter is displayed in parentheses.  In this example I wanted to find a lentiviral plasmid to express...Starting with two search terms we see there are plenty of results, but I can’t browse through 300+ plasmids... plasmids that may be difficult to define for a simple search. These pages provide helpful information...

  7. Tips for arabidopsis transformation

    Type
    Blog Post
    ... of genes and proteins in living plants, to complementing mutant phenotypes. Arabidopsis is amenable to... sweet potatoes (Kyndt et al., 2015)! It sounds simple, and it pretty much is, but there are some tips...There are a bunch of great options, but here are a couple of my favorites. Plasmids from the Tsuyoshi Nakagawa...you transform early, your plants will still have plenty of time to produce seeds after you expose the flowers...for transformants. It’s very important to select multiple T1 plants from each transformation. I like to ... Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop.’, Proceedings...

  8. Transferable Skills Guide: Identifying Your Transferable Skills

    Type
    Blog Post
    ...socks, persevered through a nerve wracking commute complete with a disabled train, and now you’re sitting ...within yourself, and to demonstrate them to the people who make hiring decisions. Though having all the...to other acquired skills.” It is nice when other people point out how awesome we are, but there is also...my wheelhouse”, take a second to reflect. For example, say your boss approaches your team with a project...front of a crowd and using your charisma to give people hope for some shiny vision on the horizon. You’...these responsibilities. If you are in charge of completing many meticulous protocols or tasks, you’ve probably...

  9. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    ...arranged in a double helix. People are wrong. In reality, cellular DNA is a complex three-dimensional globule...Technique What It Locates ChIP-seq Proteins (for our example later, histones) ChiRP-seq RNA Hi-C DNA (genome-to-genome...appendix DNA is a 3D fractal). Enhancer biology is a complex and dynamic field. We’re going to focus on a tried... beyondthedish (left) and biobabel (right). People commonly think of DNA as a linear polymer of A,...Bedtools getFasta. These regions and their reverse complements were parsed for spCas9 PAM sites (NGG) and then...

  10. A Career in Grants: How To Become a Grant Professional

    Type
    Blog Post
    ...expertise on the processes needed to successfully implement, manage and close-out a grant. Grant professionals...reports are submitted and project evaluations are completed. To enter and advance the profession, you can ...  Skills such as research, collaboration across multiple disciplines, writing, project management, and ...application writing and development, project implementation, program evaluation and grant management.  ... time and resources. 3. Get a mentor, find your people Find someone who is working or has worked as a ...knowledge of the 8 core competencies through a multiple choice exam.  The bottom line on being a grants...

  11. Negotiating Work and Life: How to Find the Joy

    Type
    Blog Post
    ...to be managing. Being reminded of the seemingly simple actions we can take to help ourselves can be an...regular schedule at work. Choose a lab/job where people have regular schedules. Work 8 to 5 or 7 to 4 or...create “fake” family by forming a social circle of people in the same situation. Go to church, join a club... in the family to have to miss something – for example, say no to some of those birthday parties. Tell...print, but I actually let my daughter skip school a couple of times. Be sure to add time with your family,...time. For me, that’s been volunteer work at our temple, my kids’ school, and with professional organizations...

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

    Type
    Blog Post
    ...al., guides were considered toxic if they were depleted from the screen. This toxicity was used a proxy... the growth screen, safe-targeting guides were depleted at greater rates than their non-targeting counterparts...Additionally, the distribution of enrichment and depletion scores for safe-targeting guides more closely ...noise, of dsDNA breaks caused by CRISPR. See supplemental figure 6 for more details. Safe-targeting guides...However, there was no significant difference in depletion of ricin sensitizer genes identified with short...Resources Watch Dr. Michael Bassik’s lecture on “Multiplexing with CRISPR Screens”    ...

  13. Viral Vectors 101: Pseudotyping

    Type
    Blog Post
    ...mechanisms behind the cytotoxicity can vary. For example, the popular VSV-G envelope protein causes cells...are differentially inactivated by nonspecific complement mechanisms in serum. Sometimes this is due to...cells that the viral vectors were produced in. For example, production of vesicular stomatitis virus, HIV-...sugars end up in the envelope and are targets for complement-based killing by anti-alphaGal antibodies. The...good at infecting most cells, but not all. For example, LDLR expression in hematopoietic stem cells, T... as described above and is inactivated by the complement system in serum (DePolo et al., 2000). Other ...

  14. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    ...Its RNA-guided nature makes CRISPRa much simpler to implement in genome-wide screens over cDNA libraries... Rochester Medical Center. During development, complex genetic programs specify and assemble diverse arrays...individual genes can be relatively laborious because multiple plasmids/viruses are needed to deliver all the... then packaged into AAV for in vivo delivery. Multiplex gene targeting is also possible and is only constrained...vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR–dCas9-activator...

  15. Changing Labor Laws Bring Increased Postdoc Wages

    Type
    Blog Post
    ...applying to, are doing - for example, your benefits may be changing. Please also feel free to contact us... salary was set at $47,476 and the date for implementation was set for Dec 1 2016. In order to be exempted...submitted letters to the Department of Labor (for example here and here) pushing for exemptions for postdocs...be on higher salaries (which have been made by multiple organizations across academia). Increased postdoc...wide consensus of academic stakeholders. There was ample opportunity to accept these recommendations, and...

  16. Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

    Type
    Blog Post
    ...combinations. For example, with standard fluorescent microscope filter sets, users can label multiple proteins ...often have absolute position requirements. For example, a KDEL ER retrieval sequence only functions at... even disrupt fusion protein localization. For example, the formation of inappropriate inter-chain disulfide...disulfide bonds between FPs blocks Golgi complex-localized fusions from properly exiting the ER and fluorescing... dark fluorescent proteins in your experiments, please consider the following: How can one ensure that...

  17. Tips for Using FRET in Your Experiments

    Type
    Blog Post
    ...revolutionised the way we apprehend molecular complexes and is still a very useful tool.    Like a guitar...technique, but it is very important to follow some simple rules to avoid common pitfalls and to use it at... These issues can be overcome by choosing a complementary pair of fluorophores. To maximize the FRET signal...especially when you investigate unknown molecular complexes. But it is a parameter that you have to keep in...most efficient approach for your own system. The simplest and the most popular one is the sensitised emission...photobleaching method The acceptor photobleaching method is simple but limited to a single measurement. This method...

  18. The Codon Guide to Synthetic Biology

    Type
    Blog Post
    ...foothills, I began to have conversations with many people who eagerly follow progress in synthetic biology...channels, books, podcasts, and communities to help people get started in synthetic biology. While not everything...NCI, NIH | License   I’m updating it regularly as people send me suggestions. The most useful parts of the...biology can be difficult to find. I hope that many people will find this guide useful as they start their...have additional resources that might be helpful, please reach out to me by email.  About the author Niko...

  19. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
    Blog Post
    ...across multiple plasmids. For simplicity, we’ll use the second-generation system as an example.  The ...Lentiviral vectors have been a staple in molecular biology for over three decades. Widely used across...double-stranded DNA transgene is incorporated into a large complex of both viral and cellular proteins and trafficked...the delivered payload is gradually reduced. For example, after using CRISPR/Cas9 to edit a gene, the expression...performing quantitative PCR titration of vector DNA in samples harvested 24 hours post-transduction (Wanisch and...

  20. Treating Muscular Dystrophy with CRISPR Gene Editing

    Type
    Blog Post
    ...hotspot for dystrophin, exons 45-55, there are multiple common deletions that maintain the protein’s reading...regularly. Dystrophin and genome editing Since complex oligonucleotide treatment comes with many challenges...skipping? For such a therapy to be successful, multiple requirements must be met. First, CRISPR would ...dystrophy. gRNAs can be designed to flank a single or multiple exons that, when deleted through NHEJ, will restore...therapy, it’s important to realize that DMD is a simpler case than other genetic diseases we’d like to treat...Thakore PI, Majoros WH, Reddy TE, Gersbach CA. Multiplex CRISPR/Cas9-based genome editing for correction...
Showing: 481 - 500 of 1010 results