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Showing: 361 - 390 of 921 results

  1. Viral Vectors 101: Transductions

    Type
    Blog Post
    ...are similar to transfections, processes which introduce foreign nucleic acid into cells by any means, ...safety considerations, they aren't dangerous for controlled lab usage. Using viral transductions can offer...selected to perform the transduction with: either retrovirus, lentivirus, adenovirus, or AAV. Then, your desired...integrate into the host genome (lentivirus and retrovirus) while others are non-integrating (adenovirus... Susanna Stroik...

  2. Three Tips for Preventing Viral Plasmid Recombination in Your Samples

    Type
    Blog Post
    ...recombination of bacterial plasmid DNA: electron microscopic analysis of in vitro intramolecular recombination. ...One of the most frequent problems that I help troubleshoot involves viral vectors and plasmids with repeating...testing your prep can save you the headache of troubleshooting failed experiments. Pick the right strain While...backbone.   Ready, set, grow! Our 5 Tips for Troubleshooting Viral Transductions will help with the next...

  3. How Dry Ice Affects Viral Vector Sample pH & How to Avoid It

    Type
    Blog Post
    ... pH. We compared the dry ice-stored samples to controls that were only stored in a -80°C freezer and not...). Before thawing, all the samples were at the control pH of 7.4 as indicated by color (data not shown...thaw. We compared vented and non-vented tubes to controls that were not exposed to dry ice. Does venting...between 0 and 96 hours. We compared these samples to controls that were only stored at -80°C. We then thawed...period of at least 96 hours had the same pH as control tubes that were never stored on dry ice/exposed...

  4. Addgene Depositors Get More Citations

    Type
    Blog Post
    ...same correlation, but it wouldn’t be causal. To control for a number of differences between those papers...methodologies, or qualities of research our adjustment controls for them.   So, for example, in 2010, 10 percent...are lots of other factors that could still be controlled for. As an example, plasmid-related areas of ...see such a big difference in citations after controlling for journal. And so, while our quantitative analysis...research tools, computing performance, and randomized control trials in innovation. Visit his website at: http...

  5. Fluorescent Protein Travel Awards - Protein Variants, a Serotonin Sensor, and an Artificial Leaf Replica System

    Type
    Blog Post
    ...fluorescent protein reporter under the control of promoters that control cell cycle gene expression.  Additional...genotype the patient and rapidly test the variants in vitro against different drugs and dosages.  Michal Bernach...-up approach will allow me to create a fully controllable and reproducible set up,” Bernach explains. ...containing unstable fluorescent proteins under the control of promoters responsive to water or nutrient levels...

  6. K. phaffii: Rising to the Occasion

    Type
    Blog Post
    ... in the general research community. Phillips Petroleum Company first commercially developed K. phaffii...operates as an obligate aerobe. It’s also a methylotropic yeast, meaning it can utilize methanol as its...peroxisomes are where methanol metabolism occurs in methylotrophic organisms. K. phaffii is an excellent model...and NHEJ is an error-prone pathway frequently introducing frameshift knockout mutations. Thus, if genetic... Susanna Stroik...

  7. Multiplexed Overexpression of Regulatory Factors (MORF) collection

    Type
    Blog Post
    ...streamline identification. While the Zhang lab controlled their multiplicity of infection so that a single...disease depends on both genetic and expression control systems to mimic illness on a molecular level. ...understand a pathway, or just looking for the best astrocyte differentiation protocol! Ready to try it out...either with or without mCherry and GFP fluorescent controls. Alternatively, you can order individual plasmids...  Susanna Stroik...

  8. CRISPR 101: Multiplex Expression of gRNAs

    Type
    Blog Post
    ...you multiplex - we’ll use mammalian plasmids to introduce you to some of your potential options and cloning...transcript You can also multiplex gRNAs via a polycistronic transcript. Rather than being transcribed from...option described below, plasmids for making polycistronic gRNAs are also available from the Yang lab for...cannot be released, adding temporal and/or spatial control to the system. pSQT1313 from the Joung lab allows...Plasmids: These plasmids are similar to the Csy4 polycistronic system described above, except that they use...gRNAs are flanked by glycine tRNAs to create polycistronic glycine tRNA-gRNA (PTG) constructs. Eukaryotic...

  9. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    ...quantity and quality of your plasmid prep is by spectrophotometry. Achieving the highest-quality DNA requires...plasmid copy number   Pro tip! If you are having trouble getting sufficient plasmid DNA, you may need to...factor label Non-silica based, avoids use of chaotropic salts   What are all these other kit types...manufacturers provide specs, protocols, and quality control information, but the level of detail can vary. ...the rationale behind each step and provide troubleshooting advice. In these cases, it can be helpful to... instructions and extensive references for troubleshooting. If you need a high level of consistency between...

  10. A Quick Guide to a Career in Software Product Management

    Type
    Blog Post
    ...skills in strong relationship-building and influence. You’ll need a large deal of influence and strong relationship-building...software product management was either. Coming from a strong background of undergraduate research straight to...energized from interacting with others, and have strong communication skills to tailor your message to ... the details, but the developers have it under control. 9:30 AM--Back-to-back meetings. Meet with my boss...

  11. Expanding the Targeting Scope and Editing Efficiency of Adenine Base Editors

    Type
    Blog Post
    ...go of the DNA. ABEs are somewhat slow and need a strong Cas9 domain to “hold on” long enough to the DNA...that expresses gene III. Gene III is under the control of a T7 RNA polymerase and cannot be expressed ...the deaminase TadA-7.10 A mutagenesis plasmid controlling the phage mutation rate Phage infection results...editing speed. Created with BioRender.com. Introducing ABE8e - a fast and flexible adenine base editor... activity. To mitigate this drawback, the lab introduced an additional mutation (V106W) into TadA-8e and...

  12. AAV Q&A with Tim Miles

    Type
    Blog Post
    ... use amplicons in ITR as we have found this to strongly bias titer. We are also adopting ddPCR, which ...the AAV.PHP.eb for neuron, oligodendrocyte, and astrocyte specificity and would like to get it working in...transfection efficiency when delivering these viruses via retro-orbital vs. tail vein systemic injections? A: We...difference in AAV transduction efficiency between retro-orbital versus tail vein injections.   Q10: Have...labs have tried to transfer PHP.eB's enhanced CNS tropism to other serotypes, but reproducible success remains...

  13. Back to Bacteria: CRISPR gRNA Multiplexing Using tRNAs

    Type
    Blog Post
    ...study the effect of gene knockout in vivo and in vitro, as well as to insert targeted mutations through...separated by 28 base Csy4 recognition sites from a polycistronic transcript. Although this method shortens construct...requires coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may have off-target... of gRNA cleavage.   Xie et. al constructed polycistronic glycine tRNA-gRNA genes (PTGs) in the forms ...Bacteria and viruses have illustrated the power of polycistronic gene organization, and its translation to CRISPR...

  14. Kiran Musunuru on the Newest TALEN Genome-Editing System

    Type
    Blog Post
    ...after the plasmids have been introduced by transfection or electroporation. All you do to create a protein...type of genome editing tool with which one can introduce changes into the genomes of mammalian or other... create a pair of designer proteins that will introduce a double-strand break in the genome anywhere you...double-strand breaks, there is a possibility of introducing a mutation somewhere else in the genome. It remains...

  15. Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

    Type
    Blog Post
    ...autophagy. Morphological assays Electron microscopy: Electron microscopy is a traditional method for...this is indicative of protein degradation). To control for protein degradation due to other pathways, ...at various stages of degradation. Additionally, electron microscopy analysis is time consuming. GFP-tagged...emit red and green fluorescence. After 4 hours of nitrogen starvation to induce autophagy, red but not green...

  16. RaPID Detection of RNA-protein Interactions

    Type
    Blog Post
    ...native regulatory elements. Controls for RaPID experiments RaPID negative controls include both scrambled ...occurring complexes present in the cell, it can also introduce non-physiological RNA-protein interactions due....  In as little was one minute BASU generated a strong streptavidin signal (~250-fold enrichment) when...when compared to a scrambled RNA motif control). BioID2 had no detectable signal at the same time point....

  17. Antibodies 101: Conjugation

    Type
    Blog Post
    ...fluorescence microscopy, flow cytometry, or spectrophotometry. The range of fluorophores available for antibody...capable of producing a visible signal, but it has a strong affinity for avidin and streptavidin, both of which...modifying the antibody sequence or that rely on strong protein-protein interactions with other peptides...of non-canonical amino acids or peptide tags. 5) Strong protein-protein interactions with other peptides...of antibody conjugation. But hopefully, this introduction provides a good base from which to dive deeper...

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

    Type
    Blog Post
    ...low-throughput methods such as patch-clamp electrophysiology, a technique that’s used to measure electric...selected for characterization with patch clamp electrophysiology. All thirty were functional and their measured...experiments, neurons expressing ChRgers produced stronger photocurrents at low light intensity than existing...the mice to increase their nose-poking behavior. Strong nose-poking behavior is exactly what was seen with...self-stimulation response. This result showed that ChRger2 stronger photocurrent properties make it better suited ...

  19. All in a Twist: dsRNA

    Type
    Blog Post
    ...If you know the sequence of a virus, you could introduce some of its dsRNA intermediates into a healthy...the inflammatory response associated with the introduction of dsRNA. Synthetic dsRNA Researchers have...time, even before we knew about all the diverse in vitro production mechanisms. siRNA, also known as RNAi...and repetitive regions, such as telomeres and centromeres. These genomic regions are prone to persistent...  Susanna Stroik...

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

    Type
    Blog Post
    ...biology - even a piece of wood - there’s a lot of control spatially as to what’s happening at different points...cellulose fibers, pattern, shape. All of that is controlled by regulatory networks in the plant. If we want... hundreds of genes and regulation and resource control - everything that’s necessary on that scale.  If... terminators. That gives us enough hardware to control hundreds of genes. We’ve created enough regulatory...transcriptional and translational activity a cell has and control how much it taxes the resources of a cell. The ...

  21. Working with Nuclear Receptors

    Type
    Blog Post
    ...reproduction. Both physiological processes are controlled by deliberate and compulsory actions instigated...corticosteroids, progesterone, androgens, and estrogens), fat-soluble vitamins A and D, thyroid hormone...complex can serve as a rate-limiting conduit to control the actions of the whole complex.  For example,...adipogenesis and macrophage activation. Our mass spectrometry-affinity purification proteomic profiling of...N. J.and O'Malley, B. W. (2002). Combinatorial control of gene expression by nuclear receptors and coregulators...

  22. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    ...derived from the E. coli Ec48 retron RT and the S. pombe Tf1 retrotransposon RT, respectively. Their small...less than 10%. In contrast, the original paper introducing prime editing demonstrated ~20-50% efficiency...fold (Anzalone et al., 2019). PE3, as originally introduced, relies on the PE2 enzyme. PE4 and PE5: Engineering...ligases to act first. PE4 and PE5, as originally introduced, rely on the PE2 enzyme. PEmax: Optimizing prime...found that the mutations that emerged depended strongly on the target edit (Doman et al., 2023). Therefore...

  23. Pathways Over Time Plasmids Engage Students in Functional Genomics Research

    Type
    Blog Post
    ...could provide the basis for student projects in introductory labs. Genome projects use computational methods... of MET/Met genes from the pYES2.1 plasmid is controlled by the GAL1 promoter. The cloning procedure adds...absence of methionine. Since the GAL1 promoter controls MET/Met gene expression, complementation should...PMC232622. 6. Winzeler, D.A., Shoemaker, D.D., Astromoff, A., Liang, H., Anderson, K., Andre, B., Bnangham...over time: Functional genomics research in an introductory laboratory course. CBE-Life Sci. Educ. 17, ar1...

  24. Antibodies 101: Stripping and Reprobing Western Blots

    Type
    Blog Post
    ...better than a nitrocellulose membrane. If you have already run your blot using nitrocellulose, you may decide...isn’t too different, consider antibody affinity: stronger binding antibodies will require harsher stripping...instructions for the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed with caution...depend on your blot — antibodies that bind more strongly or that have saturated the blot may need longer...

  25. Hot Plasmids - October 2020

    Type
    Blog Post
    ...Tsang Fluorescent proteins (FPs) traditionally introduced in Caenorhabditis elegans on extrachromosomal...gene into the target gene’s native locus, but introducing large genetic changes in C. elegans with CRISPR... donors (ssODN). So how can you use CRISPR to introduce large FP genes into C. elegans? By using a split...protein. They used CRISPR with a ssODN template to introduce the small wrmScarlet11 which is less than 60 nt...exclusively in huge bacteriophage genomes. It’s active in vitro and in human and plant cells. Its small size offers...

  26. Hot Plasmids February 2024

    Type
    Blog Post
    ...systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates. Nature Communications...Peptide-assisted genome editing for primary cells By Susanna Stroik Even with the advances in CRISPR technology over...difficult to genetically edit due to toxicity from electroporation or poor cellular entry with other transfection...viral vector has brain endothelial cell-specific tropism, with slightly reduced efficacy compared to AA9...

  27. Adeno Associated Virus (AAV) for Cell and Gene Therapy

    Type
    Blog Post
    ... disease. To do so, their T-cells were grown in vitro, their mutations corrected, and the T-cells were... body and thereby correct the mutations using retroviral vectors. However, many diseases require techniques... B, LPL deficiency, Cystic fibrosis, Muscular Dystrophy, Parkinson’s disease, and HIV. A notable landmark...infect target tissues. Improved specificity through control of gene expression and the selection of specific...CRISPR-AAV vectors Learn about Addgene AAV Quality Control Process Resources on Addgene.org Find ready-to-use...

  28. Tips for Titering Your Lentiviral Preps

    Type
    Blog Post
    ...method can be the absence of suitable controls. The best control for this type of assay is a clonal cell...using a reverse transcriptase mutant in a separate control reaction. This will allow you to quantify the amount...of the qPCR target gene. Developing the proper control line typically requires transducing with a limiting.... 3. Zhang, Bing, et al. "The significance of controlled conditions in lentiviral vector titration and...

  29. Experimenting in an Open Source lab: from CRISPR to Cats

    Type
    Blog Post
    ... our members took an Arduino microcontroller and a motor controller and made it spin again!  We’ve also...scientists can get involved in modelling, the electronics folks can build a robot worm on top of that model...instance, the centrifuge in the picture had a broken controller and would not spin. It was going to cost far ...far more than it was worth to repair the controller and a new replacement was not possible, but one of our...

  30. Adenoviral Delivery of CRISPR/Cas9 Aims to Expand Genome Editing to Primary Cells

    Type
    Blog Post
    ... mutated gene. In a Scientific Reports paper introducing the delivery method in 2014, Manuel Goncalves...include their episomal nature and very efficient introduction of DNA into therapeutically relevant, non-transformed...B-thalassemia, Sickle Cells Disease, Duchenne Muscular Dystrophy, Alpha1-antitrypsin deficiency (most widely used... for cancer therapy. Elsevier, pp 39–67 Ding Q, Strong A, Patel KM, Ng S-L, Gosis BS, Regan SN, Cowan ...adenoviral delivery of CRISPR/Cas9. Journal of Controlled Release 298:128–141 . https://doi.org/10.1016...
Showing: 361 - 390 of 921 results