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We narrowed to 850 results for: met

Showing: 301 - 320 of 850 results

  1. CRISPR 101: Off-Target Effects

    Type
    Blog Post
    ...nucleases are promiscuous. In other words, they can sometimes localize to unintended sites with sequence similarity...In this instance, a 5% off-target frequency is something to keep in mind, but likely will not have a significant...many applications, it is the only comprehensive method available. Targeted sequencing approaches More...2023) for a review of other targeted sequencing methods. Candidate site sequencing Sites with high sequence...comprehensive approach, RNA-seq would be an appropriate method for an activator or repressor Cas. Similarly, Cas...suitable. How to control for off-target events Sometimes there just isn’t a way around off-targets. Your...

  2. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    ...because phages frequently alter their DNA by adding methyl groups, glycosyl groups, and other modifications...post, I will describe a useful and cost-effective method that our lab (the Szymanski lab, which has recently...examples of RBPs successfully identified using this method. Step 1: Prepare a random fragment library of phage...nitrogen gas tank with a pressure regulator. Other methods for physical shearing exist as well, such as sonication...sonication and needle shearing. Essentially, any method that results in the bulk of resultant fragments...library construction for functional genomic and metagenomic screening." Applied and environmental microbiology...

  3. What's New in CRISPR - September 2019

    Type
    Blog Post
    ... from an analysis of human metastasis cohorts and a prior mouse metastasis CRISPR screen. The Chen Mouse...lab discovered a new Cas protein, CasX, from metagenomic analysis of microbial DNA from groundwater. Now...knockout libraries for cancer research. The Mouse Metastasis CRISPR-Cpf1(Cas12a) Double Knockout Library is...tumor suppressor genes (TSG). Find the Mouse Metastasis CRISPR-Cpf1 Double Knockout Library Find the...

  4. Important Considerations When Using AAVs

    Type
    Blog Post
    ... Serotypes and Tropism Experiment  Detection Method CNS AAV1, AAV2, AAV4, AAV5, AAV8, AAV9 Heart...neuron specific transfection using Cre-lox, this new method could allow for labeling of neurons showing a very...very specific phenotype. Previous neural tracing methods used viruses that were potentially toxic and would...same cell types. Not only can we compare these methods in specific applications, we may also be able to...understanding of receptor physiology and the effects of metabotropic and ionotropic influences on overall cell function... genetic engineering." Journal of neuroscience methods 235 (2014): 331-348. PubMed PMID: 25107853. 12....

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

    Type
    Blog Post
    ... sites. The lambda red system is an alternative method that can be used for cloning or genome engineering...50% by designing oligos that avoid activating the methyl -directed mismatch repair (MMR) system. MMR’s job... beta and gam.  A major advantage of using this method for lambda red expression is that it doesn’t require...biology technique. However, an advantage of this method is that you can stably integrate the Red system...Recombineering: A Homologous Recombination-Based Method of Genetic Engineering. Nature protocols. 2009;...Plasmids to Disrupt Genes in Mammalian Cells CRISPR Methods for Bacterial Genome Engineering Resources on ...

  6. Plasmids 101: Gene, enhancer, and promoter trapping

    Type
    Blog Post
    ...dispensable or are the current identification methods missing something? Here we will review some time-tested ...reporter construct can be something phenotypic (such as a fluorescent protein or something species-specific) ... one another. Under these circumstances, trap methods may be the preferred screening option. Promoter...

  7. New CRISPR Web Resources and #12DaysofCRISPR Recap

    Type
    Blog Post
    ...histone demethylation by LSD1, cytosine methylation by DNMT3A or MQ1, and cytosine demethylation by Tet1... Delivery Methods and A Match Made in Heaven: CRISPR and AAV Day 6: Bacteria: CRISPR Methods for Bacterial...

  8. Viral Vectors 101: Transductions

    Type
    Blog Post
    ...usually bacterial in nature. All three of these methods involve DNA incorporation into the cell, but the...next experiment. Using transduction vs. another method Transfection and transduction seem to achieve similar...these questions will help guide you to the right method for your experiment.  A cool feature of viral work...picky when it comes to transfection/transduction methods. Some ‘difficult to transfect’ cells lines are ...which can’t be easily achieved with alternative methods. Ready, set, transduce! Making virus can sound ...

  9. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

    Type
    Blog Post
    ... PA-FP for your experiment, there are several parameters to consider, such as brightness, photostability...leading to protein aggregation. Another important parameter to consider is the contrast ratio- a comparison... PA-FPs have helped create several new imaging methods, including photoactivation-localization microscopy...high-resolution two-color fluorescence microscopy." Nature methods 6.2 (2009): 153-159. PubMed PMID:19169259. PubMed...photoactivatable fluorescent proteins for CLEM." Nature methods 12.3 (2015): 215-218. PubMed PMID:25581799. PubMed...indicators for targeted neuronal imaging." Nature methods 12.9 (2015): 852-858. PubMed PMID:26167640. PubMed...

  10. Cancer and the Immune System: Deciphering the Relationship

    Type
    Blog Post
    ...creating tolerance, and evading recognition are a few methods by which tumors sidestep immune surveillance: Suppressing... tumor cells spreading to other organs during metastasis. Evading Recognition Tumor cells over-express...damage healthy cells, and often cannot eradicate metastasis or eliminate the probability of a relapse. Chemical...the immune cells that aid tumor formation and metastasis, block the cell-surface proteins that help escape...specific antigens to activate them are a few methodologies being researched. Apoptotic and necroptotic ...localized tumors or systemic tumors), and ability to metastasize. There have been reports of clinical trials going...

  11. Plasmids 101: Screens vs. Selections

    Type
    Blog Post
    ...first question is: to screen or to select? Which method will work best for your scientific question? If...Why the confusion? The commonality between these methods is that both attempt to identify new players in...those factors is assessed. The two most common methods of manipulation are genetic knockout mutations ...Thus, a limited panel is usually selected. The method you choose to assess traits of interest is highly...cells and thus not assayable with CRISPR. Another method, popularized in yeast, are temperature sensitive...

  12. Hot Plasmids - May 2022

    Type
    Blog Post
    ...expression using Cre-mediated AAV Targeted Evolution method (M-CREATE) dCas9-SSAP with reduced error rate for...expression using Cre-mediated AAV Targeted Evolution method (M-CREATE) by: Brian O'Neill  The Viviana Gradinaru...version of the Cre-mediated AAV Targeted Evolution method (M-CREATE), in which one applies both positive ... by: Lucie Wilson Le Cong’s lab has developed a method using dCas9-SSAP to knock-in long sequences with...to 20%, comparable to commonly used CRISPR/Cas9 methods for large inserts. However, this system has a much...

  13. AAV Q&A with Tim Miles

    Type
    Blog Post
    ...in sample treatment across methods can affect reported titer by either method so to ensure equal animal...vector, split intein approach.   Q3: What’s the best method for titering AAV? Is there a difference in titer...traditionally used qPCR (protocol link). We have found this method to be reliable and reproducible. However, we do...animal dosing, we advise that users keep titering method consistent across samples within a project. Targeting...

  14. Viral Vectors 101: Inverted Terminal Repeats

    Type
    Blog Post
    ...with biorender.com. ITR structure  WT ITRs are symmetrical 145-nucleotide sequences that flank the ends ...not properly assemble, but these sequences can sometimes be removed to improve vector stability and sequencing...mutations will go undetected by restriction digest methods.    Resources and references More resources on...the Baculovirus/Sf9 Cells System. Hum Gene Ther Methods, 28(5), 277–289. https://doi.org/10.1089/hgtb.2016.133...Repeats of Adeno-Associated Virus. Hum Gene Ther Methods, 30(6), 206–213. https://doi.org/10.1089/hgtb.2019.276...

  15. Multiplex Genome Editing with CRISPR-Cpf1

    Type
    Blog Post
    ...this new Cpf1 multiplexing method, other multiplex CRISPR gene editing methods relied solely on Cas9. Overall...Cas9 Multiplexing Options  Multiplexing Method Delivery Method Advantages Disadvantages References Yamamoto...CRISPR technologies.  It’s a simple and effective method with multiple applications (in vitro, in vivo, ...

  16. Screening for Successful Genome Editing with Digital PCR

    Type
    Blog Post
    ...your favourite gene, there are several different methods available. Thankfully, there are many great resources...® or T7E1) for mutation detection. While these methods paved the way for mutation validation, we found...workflow. Digital PCR comparison to other validation methods As mentioned, these assays have several major advantages...human iPS cells without antibiotic selection. Nat Meth 11, 291–293 (2014). PubMed PMID: 24509632. PubMed...Berman, J. R. & Postovit, L.-M. A Digital PCR-Based Method for Efficient and Highly Specific Screening of ...

  17. 3 Challenges in Plant Synthetic Biology

    Type
    Blog Post
    ...not understand, and the complicated and unique metabolic processes of plants are being investigated on ...biochemical pathways involved in central anthocyanin metabolism are well understood. Additional work has also...knockouts to better understand developmental, metabolic, and photosynthetic processes. Often the tools...like a lot of specialized science, these tools sometimes have limited distribution. We hope that will change...more difficult by the fact that transformation methods have also been patented. In order to construct ...

  18. Channelrhodopsins with improved light sensitivity for minimally-invasive optogenetics

    Type
    Blog Post
    ...Optogenetics is a neuroscience method that lets you fire neurons with the flick of a light switch. Neurons...channelrhodopsins with high-throughput proteins engineering methods, like directed evolution, because channelrhodopsins...channelrhodopsins are typically evaluated with low-throughput methods such as patch-clamp electrophysiology, a technique... was delivered and activated with non-invasive methods to the neurons in the right secondary motor cortex...engineering enables minimally invasive optogenetics. Nat Methods 16:1176–1184 . https://doi.org/10.1038/s41592-019...

  19. Giving gRNAs a Facelift - Synthetic and Beyond

    Type
    Blog Post
    ... of the most common of these is 2’-O-methylation, where a methyl group is added to the 2’ hydroxyl group...protect the RNA from degradation, several simple methods have been employed which modify the sugar or phosphate...together, for example phosphorothioates with 2’-O-methyl modifications, proved more stability than just ...similar advantages, bridged nucleic acids with N-methyl substitutions are slightly more efficient and less...

  20. Antibodies 101: Selecting the Right Antibody

    Type
    Blog Post
    ...which may contain more detailed data on the assay methods. You might find that while the website highlights...direct (primary-conjugated) or indirect (secondary) methods. In those cases, remember that a secondary should...anti-tag antibody; and immunoprecipitation-mass spectrometry strategies that isolate an antibody’s immune...complexes and confirm interacting partners by mass spectrometry (Uhlen, 2016). Be sure that the antibody you...) A proposal for validation of antibodies. Nat Methods 13:823–827...
Showing: 301 - 320 of 850 results