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We narrowed to 1,025 results for: URE

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  1. Filming Science Videos in the Age of Social Distancing

    Type
    Blog Post
    ... adjustments I wanted to make to ensure smooth filming in the future, but we were finally refining the...I could be happy with it. Cycle 5 - The Future! Figure 5: The mobile monitor and teleprompter being...trying all over again. Here’s that process!   Figure 1: The Design-Build-Test-Learn cycle adapted for...anticipated, we had to think of something else.   Figure 2: Ironing boards. Good as a WFH desk, and as a...important for me to let people record at their leisure. For virtual B-Roll (that’s the secondary footage...drawing board.  Cycle 3: A Return to HQ Figure 3: Even Blugene is practicing social distancing... left the room. Microphone: Lastly, I needed to figure out a microphone situation. Generally, I record...

  2. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    ...extension can usually ensure that the 3’-base(s) do not form secondary structures. The introduction (or...overlap ensures that the PCR generates a nicked circle rather than a linear product (see figure). Template... simply following the polymerase manufacturers’ PCR protocol. Be sure to adjust the extension time to ...vivo to give the final circular plasmid product pictured here. B) Screening of recovered plasmids for the...final concentration). The DMSO reduces secondary structures of the DNA template, and may also decrease the...template for site directed mutagenesis.  Polymerase To ensure that you don’t introduce undesired mutations through...polymerases such as Taq generate A-overhangs (a feature used in TA-cloning). Such non-complementary bases...

  3. Treating Muscular Dystrophy with CRISPR Gene Editing

    Type
    Blog Post
    ...Staphylococcus aureus Cas9. Nature. 2015 Apr 9;520(7546);186-91. doi: 10.1038/nature14299. Epub 2015 Apr...some research in human embryos. Nature. 2015 Dec 3. doi:10.1038/nature.2015.18947 5. Siva K, Covello G... mouse model, an important proof of concept for future preclinical and clinical work. Find Plasmids for... is of course much more difficult than in cell culture, but Ran et al. have shown that CRISPR and AAV ...homology-directed repair (HDR) pathway is downregulated in mature tissues - no problem, as exon skipping proceeds...would be applicable to 80% of DMD patients. Figure 1: Exon skipping strategies for Duchenne muscular...low-frequency indels will not cause frameshifts. Figure adapted from Osterout et al. (2015b) under a CC-BY...

  4. How to Negotiate a Successful Lab Start Up Budget

    Type
    Blog Post
    ... like pipets, or mini-centrifuges, etc. Be sure to capture as many of these tools as possible, this even... we suggest estimating the time it will take to secure your first (R-level) research grant.  Set your ...down prices as you create your list. This helps to ensure that all of the resources are accounted for. Write...an idea of how much you can expect to use in the future. 3. Identify the skills needed to use these tools...the talent pool that you’ll need to tap for your future research team. For example, if you’re going to ...help you define salary costs. 4. Ascribe a cost structure associated to these tools, skills, and personnel...searching for a faculty position. This is important to ensure that you’re looking at institutes that have the...

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

    Type
    Blog Post
    ...action potential (Figure 1). This light-induced excitation can take place in a tissue culture dish but can ... which often forces scientists to use invasive measures to perform in vivo optogenetic experiments. Intracranial...implantation of fiber-optic cables into the brain. Figure 1: With no blue light, the channelrhodopsin remains...improved photocurrent properties: The sequence and structures that determine membrane expression and plasma... electrophysiology, a technique that’s used to measure electric current of ion channels in living cells...electrophysiology. All thirty were functional and their measured photocurrent amplitude, wavelength sensitivity...were then characterized further by expression in cultured neurons and mouse brain slices collected following...

  6. Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9

    Type
    Blog Post
    ... Design and testing of eSpCas9 Looking at the structure of the Cas9 nuclease (PDB ID: 4OO8 and 4UN3), ...Interactions between the target strand and the gRNA (see figure below) Because off-target sequences have less ...theoretically decrease off-target cutting. Figure 1: Strand separation and target DNA binding to ...improve the specificity of Cas9 derived from S. aureus. This means you can likely apply similar rational...of genomic sites. Knowledge of Cas9’s dynamic structure leads to the development of HypaCas9 Researchers..., Chen et al. 2017 used single molecule FRET measurements to determine which portions of Cas9 might be...HypaCas9 plasmids are available from Addgene here! Future possibilities for Cas proteins With their enhanced...

  7. A Control for All Seasons

    Type
    Blog Post
    ...experiments require positive and negative controls to ensure proper interpretation of results. A positive control... to compare the experimental group.     Figure 1: A, Most biological experiments will benefit ...group and C, technical replicates or repeated measurements will increase the power of the results. In this...experiment. Technical replicates are repeated measurements of the same sample and are included to assess...long as the protocol remains the same.    Figure 2: Microscopy-based antibody experiments should...molecules through the interactions of other antibody structures, such as the Fc region. Fc-mediated binding interactions...as with an isotype-matched control.      Figure 3: A preimmune serum control addresses situations...

  8. Plasmids 101: Dimers and Multimers

    Type
    Blog Post
    ...bacterial culture into the late stationary phase. Growing the culture at a lower temperature such as 30...  Figure 3. Summary of plasmid multimerization troubleshooting tips. Portions of this figure were ... More research is ongoing to determine plasmid features that affect the frequency of multimerization. .... Theoretically, as long as all the plasmid features remain intact, plasmid multimers function just as...recA-independent mechanisms. Avoid overgrowing the bacterial culture Another way to prevent plasmid multimerization ...optimizing the incubation time can help maintain cultures in log phase growth. In a study by Williams et...plasmid can exist in a bacterial population as a mixture of monomers and various forms of multimers (Summers...

  9. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    ...p300-P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks to histones in...plasmid form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters...also available as ready-to-use lentivirus. Figure 3: dCas9-DNMT3A methylates cytosines at promoters...LSD1 to the smaller nuclease-dead Staphylococcus aureus Cas9 (Sa-Cas) and deposited plasmid IF311: pMAGIC...silencing was transient and quickly reversed in culture. However, DNMT3A-induced methylation persisted ...period, as this mark was faithfully propagated in culture and in vivo. In certain cases, epigenetic modifiers...acetyltransferase activates genes from promoters and enhancers. Nature Biotechnology 33:510–517 . https://doi.org/10.1038...

  10. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    ...polysaccharide structures, many of which are easily varied by the cell and whose structures are just beginning...around 8-12 h at room temperature as opposed to overnight at optimal growth temperature. If you’re lucky enough...traction lately (1, 2), depends on knowledge of the structure(s) a given phage will recognize on a host cell... for by manipulating the nebulization time and pressure. Nebulization units, which can be purchased cheaply...attached with a hose to a nitrogen gas tank with a pressure regulator. Other methods for physical shearing...some clones being outcompeted by others in liquid culture, dilute and plate the complete library of transformants...paper (colony-side up) and leave for 1 h at room temperature. After this, it is helpful to subject your membrane...

  11. Finding Your Perfect Job After University

    Type
    Blog Post
    ...everything was more structured and controlled. I worked from Standard Operating Procedures (SOP’s) and every...perfect duration for me because it was sufficient to figure out if my image of lab work lived up to reality..., but in research you also experience a lot of failure and problem solving. I believe that is something...they never really prepare you for the amount of failure and problem solving scientists experience during...in ways that people from another background or culture could understand. I did, however, find it very ...learn how to cope with a different climate and culture, and also needed to be resourceful and adapt. By...doing experiments  without the constant fear of failure because I knew the tests worked and would produce...

  12. Meet an Addgenie: Insights into Grad School, Career Paths, and More

    Type
    Blog Post
    ...My name is Lauren Flynn and I am currently wrapping up my co-op with Addgene as a DNA Sequencing Analyst... member of the Quality Control team, I help make sure the plasmids that go out our doors are correct. ...minor, it’s time for me to start considering my future path. Talking to Addgenies has helped me get a ...concepts that are heard often during undergrad. Lauren Flynn is the Addgenie behind our "Meet an Addgenie...years ago at a CRISPR meeting.Read his Instagram feature here. You also don’t have to go back to school...that if you don’t get your PhD you’re set up for failure. Listening to Kenkel’s story has helped me realize...with our resident dinosaur! Read her Instagram feature here. One of our QC Scientists, Shreya Vedantam...

  13. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    ...through the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed...difficult to target specifically with a gRNA). Be sure to keep these techniques in mind when choosing a... to knock out exon 2 of a given gene.   Figure 1: A knockout targeting vector designed to insert...for targeting constructs. In the example shown in Figure 1, large sequences corresponding to exons 1 and...-eGFP-PGK-Puro from the Jaenisch lab.   Figure 3: A knockout/knock-in targeting vector designed... and the targeted exon in the gene of interest (Figure 4). When recombination occurs, the gene still functions...cassette has been placed into an intron.    Figure 4: A targeting vector to create a floxed allele...

  14. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    ...long dsRNA via an RNase III enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire...involved in the biogenesis of different small RNAs (Figure 1). These siRNAs can exert their gene silencing... modifications of complementary gene sequences (Figure 1). As a result, gene silencing can be easily induced...from the ease of use in dsRNA applications. Figure 1: Schematic of an RNAi-inducing transgene. Typically...that facilitates the formation of a hairpin RNA structure, resulting in processing by the endogenous RNAi...greatest advantage of RNAi is the straightforward nature of the technique—all that is required dsRNA complementary...complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes can be ...

  15. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    ...vectors effectively. Retroviral structure   Figure 2: Structure of a retrovirus. Image re-used ... you can use Figure 1 to compare the genomes of these two family members.   Figure 1: Gamma-retrovirus...2021 under CC-BY license. Figure 2 gives a detailed view of the structure of a retroviral particle, which...   Figure 3: The life cycle of a retrovirus. Image courtesy of Jeremy Luban, MD. Figure 3 depicts...vectors correctly. A solid understanding of the structure of a wild-type viral particle — and of the normal...depicts the viral life cycle. One should ponder this figure often, especially the three steps from the 12 o...

  16. MXS Chaining

    Type
    Blog Post
    ...fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave researchers the...various mammalian promoters), the same cloning procedure can be used to incorporate each element into the...These fluorescent reporters can be used to detect structure and protein localization, as well as cellular ...with MXS-chaining were used to label subcellular structures in live cells. Four individual expression constructs...fluorophore, and thus enabled labeling of particular structures (Table 1). Each construct was flanked with a ...final construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling...Table 1: MXS Constructs for Labeling Cellular Structures Construct Fluorophore Excitation/emission...

  17. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    ...bacterial recombination pathway to allow any cloning procedure to be performed using a simple two-step, 2 hr ...hr protocol prior to transformation (Figure 1). As this approach requires no special reagents or purification...be immediately adopted by any laboratory. All procedures are performed using the same three step protocol...cells. Primer design Using specific primer design (Figure 2), IVA cloning can be used to perform any plasmid...design for each type of modification is shown in Figure 2, and is discussed in more detail below. The main... the template binding region, with a melting temperature (Tm) of around 60 °C for all primers. The length...assembly. Combining multiple modifications A key feature of IVA cloning is the ability to combine multiple...

  18. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

    Type
    Blog Post
    ...halt degradative processes while maintaining the structure of your samples, the relationships between cellular...that occurs depends on the incubation time and temperature, so you may be able to recover good antibody ...cross-linking, but it doesn’t preserve sample structure quite as well as a cross-linker. This is because...fixation with these chemicals, which impacts sample structure and could also cause your target to be washed ...Furthermore, these chemicals can also alter the structure of proteins, which could disrupt your target epitope...good thing depending on your experiment.   Figure 1: Choice of fixation method impacts antibody binding...dehydrates the protein, which can disrupt protein structure and thus structural epitopes, but can reveal linear...

  19. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ...fusion proteins can visualize: Cell Structure Plasmids: Cell Structure Plasmids: ER mCherry-Sec61 ...domain. Feng Zhang's group utilized the bi-lobed architecture of Cas9 to engineer a series of "split" cas9...for iLID nano and 800nM to 47µM for iLID micro; Figure 1). With this great affinity range, these LIDs ...light-mediated subcellular localization in mammalian cell culture and reversible control of signalling pathways. ... off and on your favourite signalling pathways (Figure 2). They are all available now at Addgene. 1...efficiency and gene expression. The pUCXKT vector ensures that all screened plasmids contain a gene variant....g. the ER) and a reporter protein of interest (Figure A). Franck Perez’s lab has engineered hook proteins...

  20. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    ...required longer exposure time than its protein equivalent and was less stable due its RNA nature, it paved ...including: Environments with higher temperatures, since proteins can denature while aptamers are more stable...form the base of the stem in an RNA stem-loop structure (see Fig. 1a). When the aptamer binds its ligand...laboratory took advantage of their deep knowledge of the nature of GFP fluorescence to create an improved fluorescent...fluorescent aptamer system. Knowing that GFP’s  structure stabilizes a 4-hydroxybenzylidene imidazolinone...cells. The new aptamer also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium... its biosensor capabilities (see Fig. 1b). The future of aptamer fluorophores The Jaffrey lab and others...

  21. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    ...Zhang, a Ph.D. candidate in the Yang Lab, Qi Li, a lecturer at Sichuan Normal University, Jieze Zhang, an ...plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing, an arabinose... now take place. After editing, pTargetF can be cured from the cell by inducing expression of the sgRNA...other edits. Once you have all the edits you want, cure pCas9 by growing the cells at 37 °C.     The new...us to update the pCas/pTargetF system.   Figure 1: The editing process begins with the addition...DNA for editing to take place. Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously..., 2) changed the replicon of pCas into a non-temperature-sensitive replicon, and 3) added the sacB gene...

  22. CRISPR 101: Targeting RNA with Cas13a (C2c2)

    Type
    Blog Post
    ...toolbox. We'll cover how Cas13a was identified, the structure and function of Cas13a with a focus on what makes...genes that fell into 3 categories based on the architecture of the CRISPR protein in question: C2c1, C2c2... referred to as Cas13a) is unique in terms of structure and function, and will therefore be the focus ...cleaves RNA rather than DNA substrates. In terms of structure, Cas13a shares no homology to the most commonly...CRISPR Blog Posts Applications of Cas13a   Figure 1: Using Cas13a as a diagnostic tool. A pool of...applications of Cas13a given what we know about its structure and function? For starters, Cas13a can be used...2017). This system, dubbed SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika...

  23. Multifunctional Neural Fibers: Modulation from Gut to Brain

    Type
    Blog Post
    .... These features enable the fibers to emit light for optogenetic purposes, measure temperature, study ... described, then measured electrophysiological potential afterwards. This measurement trended with expression... the fibers would need to be, as well as other features necessary for implantation in brain tissue. The...the wild west for fiber implants. Due to the soft nature of the intestines and the requirement for digestive...multiple channels and can also record tissue temperature proximal to the implanted fibers. As important...Alternatively, the system could be used to deliver or measure new variables. The sky is really the limit for ...wireless modulation of gut and brain neural circuits. Nature Biotechnology, 1–13. https://doi.org/10.1038/s41587...

  24. Trading Spaces - Visiting Scientist Edition

    Type
    Blog Post
    ...prior to your departure for the new placement. Make sure you read any relevant literature for the techniques... have structured visiting positions within defined labs which you can apply to. These structured types...institute and mentor that they may want to consider for future work placements. The chance to learn and collaborate...prior to arrival so that you can work together to ensure your expectations are reasonable, your reagents...Unless some of these logistics are covered by a structured visiting program, you will have to make arrangements...get the ball rolling! If you are applying to a structured program, they likely have guidelines to consult...don’t make your bench mentor wait on you!), make sure you are prepared (read any protocols the day before...

  25. Plasmids 101: NGS Quality Control for Pooled Libraries

    Type
    Blog Post
    ...amplification and verification processes we use to ensure high-quality library distribution. You can also...library contains many plasmids, care must be taken to ensure that all of these plasmids are amplified and none...First, use enough DNA and competent bacteria to ensure all components of the library are represented. ...performing amplifications, and we use electroporation to ensure a good transformation efficiency. For a typical...Addgene still recommends plating the transformation mixture on solid media. The colonies are then scraped off...we created for the Brunello library is shown in Figure 2. You can use a small PCR product because the ...AUC of 0.5 representing perfect uniformity (see Figure 3A). Addgene finds that this representation is ...

  26. Viral Vectors 101: Inverted Terminal Repeats

    Type
    Blog Post
    ...(rAAV) vector (Figure 1). Everything else outside the ITRs gets left behind.    Figure 1: Recombinant...polyA signal. Created with biorender.com. ITR structure  ITRs are symmetrical 145-nucleotide sequences...palindromic arms (A-A’, B-B’, and C-C’) that give the feature its characteristic T-shape. The arrangement of ... the ITR, which can either be “flip” or “flop” (Figure 2). Additionally, the ITR contains a 4-nucleotide...target site for Rep proteins (Savvy et al. 2013). Figure 2: AAV2 wild type ITR in flip and flop orientation... B-B’, and C-C’ regions) and forms a T-shaped structure. The Rep binding element (RBE) and terminal resolution...FAQs Due to their high GC content and hairpin structures, ITRs are prone to replication errors and disruptive...

  27. Photosensitizer Induced Cell Ablation with FAP-TAP MG-2I-dL5**

    Type
    Blog Post
    ...experimental settings: HEK293 cell culture and in live zebrafish. For cell culture experiments, only dL5**-expressing...just 1 minute of exposure. MG-2I-dL5** experienced minor bleaching after longer exposure times. Additionally... fluorogen that can be used to ablate cells in culture and in vivo.  Read on to learn more about this ...exposed to light stained dead within 30 minutes of exposure. 90 seconds of illumination were sufficient for...mCer3 cells present 96 hours post treatment (see figure 2). Induced cell death was confirmed by TUNEL staining...light. They found that KillerRed required longer exposure times (5 minutes vs 10 seconds) to achieve the...genetically targetable near-infrared photosensitizer. Nature Methods,13(3), 263-268. doi:10.1038/nmeth.3735....

  28. Fluorescent Proteins 101: Luciferase

    Type
    Blog Post
    ...many available to choose from) is now a common feature in reporter plasmids.  Learn How Luciferase Can... reporter protein or its enzymatic activity is measured. The effect of promoters or enhancer regions on...case of a luciferase assay, photon emission is measured resulting from the catalysis of a chemical reaction... Green Fluorescent Protein (GFP) because of the nature of the chemical reaction compared to using a high-intensity...improved signal sensitivity since photons are simply measured – they are not required to initiate the reaction...Expression of luciferase as the reporter gene is measured via its activity to produce light, often with ...luminometer or scintillation counter, and the activity measurement can then be used to quantify the effect of the...

  29. Antibodies 101: Flow Cytometry Controls

    Type
    Blog Post
    ... conjugated antibody. Compensation beads are a mixture between synthetic beads that can bind to a conjugated... your voltage using your single color controls (Figure 1A), instead of using your actual samples. They...the settings with experimental samples. Figure 1: Example flow plots for a single color control... may otherwise be difficult to separate out. In Figure 2, you can see that, without the FMO, it would ...population from the Ly6C positive population. Figure 2: An FMO gating example. The FMO sample (A) can...clear positive and negative population. If you’re unsure what your population will look like for a particular...provided by the antibody companies or check the literature. Pro tip! Early gates, like your live/dead markers...

  30. Viral Vectors 101: Viruses as Biological Tools

    Type
    Blog Post
    ... from viruses that infect people in nature, extra safety measures, like replication incompetent viral ...the stomach flu), and many more. Their infectious nature allows them to easily deliver their “cargo” to ... make you sick…well, you feel it. But the same features that make these familiar viruses such a pain is...biological tools.   Viruses as biological tools In nature, viruses infect cells and organisms (which we refer...of cell the virus will target and fuse with. In nature, this determines which organisms and cells they...Institute. Its genome, however, isn’t enough to ensure the virus can replicate and spread. Instead, viruses...are required. We will be covering these safety measures in a separate, upcoming post. If a viral vector...

  31. Plasmids 101: Screens vs. Selections

    Type
    Blog Post
    ...yeast, are temperature sensitive screens. These involve raising or lowering the culture temperature to exacerbate...Selections do require there to be a culture condition that can apply enough pressure to select for variants of ...individually administered and then outcomes are measured. While the entire genome can be knocked out with... have a screen that doesn’t yield any hits. Make sure you plan ahead and consider these questions to save...though. A selection can take the form of varied temperature, nutrient availability, drugs, and much more....with screens. Selections require there to be a culture condition which eliminates manipulations that do...conditions which convey resistance to a drug. You could culture cells in the drug of interest at a lethal dose ...

  32. Hot Plasmids - February 2022

    Type
    Blog Post
    ...perturbation The Penn State Protein Ladder Engineered miniature CRISPR-Cas system for mammalian genome regulation...frankenbodies include mEGFP, mCherry, and HaloTag. Figure 1: (a) Anti-FLAG frankenbody (FB) construction ...advantageous to Cas9’s larger expression requirements (Figure 1), but Cas12a has generally been underutilized...finding acceptable variations in DR sequences. Figure 2: comparison of Cas 9 vs. Cas 12a expression cassettes...optimized toolkit furthers Cas12a’s potential in future genetic screens.   DeWeirdt PC, et al. Nat Biotechnol...commercial ladders which also provides a useful yet low-pressure teaching opportunity for lab members or students... to protein expression and purification. Figure 3: The Penn State Ladder run on SDS-Page gels. ...

  33. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...repository, but be sure to let us know if you'd like to write about your plasmids in a future blog post. No...under the control of the cI857 temperature-sensitive repressor. The temperature-controlled expression of the...crystallographic structures available in the PDB. Parton DL, Hanson SM, Rodríguez-Laureano L, Albanese SK...articles. These brief articles highlight the main features and applications of a partiular plasmid or set...arduous. The cloning process alone involves much literature searching, DNA synthesis, and plasmid assembly.... The pooled library, designed to be used with cultured Drosophila cells, targets 13,501 fly genes with...will create new traits in bacteria. Wang, et al. Nature. 2009. PubMed PMID: 19633652 Court, et al. Annu...

  34. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    ...and one NHEJ-repaired indel, often occur. Figure 1: In conventional gene targeting (A), you must...be included in the final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair...challenging for sequences with complex secondary structure. Today, multiple companies offer long single-stranded...directs cleavage right before the stop codon!  Figure 3: How to design a C-terminal insertion with Easi-CRISPR... To avoid re-cutting by Cas9 after editing, make sure that the repair template does not contain the wild...facing towards or away from each other.  Figure 4: How to design a conditional allele with Easi-CRISPR...you have a plasmid with your desired insertion feature (e.g., a fluorescent protein), you can amplify ...

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

    Type
    Blog Post
    ...lives of people and society. Future generations of IMP Motivating future generations is key to the success...internationalization become a reality available to all youth, our future, around the world.   In many countries of the world...improvement) and bright students that will be the future of the country. Brain drain is a serious problem...build, together with the universities, a bright future. IMP provides matched one-on-one international ...support to increase the chances of success, but also nurture a supportive and protective environment where individuals...empowered to productively navigate through both their failures and their successes. Although all mentorship relationships...their immediate frontiers. Moreover, sharing a culture/country of origin with an international mentor ...

  36. How Exercise Purges Stress and Keeps Depression at Bay

    Type
    Blog Post
    ...expression of kynurenine aminotransferases, to enhance the conversion of kynurenine into kynurenic acid. That...That’s key because, unlike kynurenine, kynurenic acid can’t cross the blood-brain barrier. By lowering...muscles led to another clue: differences in the kynurenine pathway of tryptophan degradation. It turns out...lowering circulating levels of kynurenine in the body, the brain is protected from stress-induced biochemical...brain’s physiological demands,” they write. “In the future, these may lead to treatments for a variety of ...References: Skeletal muscle PGC-1α1 modulates kynurenine metabolism and mediates resilience to stress-...

  37. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    ...pellet for the remainder of the cultures and later maxiprep only the cultures with intact ITRs. I use a spreadsheet...proper packaging of AAV, but also form secondary structures that are prone to deletion from the transfer ...this issue. One way is to grow multiple bacterial cultures of your AAV transfer plasmid at 30 °C instead ...like to miniprep 5 mL of my AAV transfer plasmid cultures and then screen with SmaI digests. I freeze the...PEI transfections. It saves me time and helps me figure out if I have enough plasmid to complete my experiment... viral particles for efficient gene transfer. I figure it’s better to have a bit extra AAV than to have... skip the PEG precipitation of the HEK293 cell culture supernatant because it’s time consuming to prepare...

  38. Hot Plasmids and Viral Preps - May 2021

    Type
    Blog Post
    ...synthetases.     Figure 2: Specific binding of scFVs to cognate aaRS antigens as measured by suspension ...encapsulating the protein of interest (Figure 3).   Figure 3: Schematic representation of the MPyV...domain (RBD) of the viral spike protein.   Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and...resolved fluorescence, and suspension bead assay (Figure 2). After finding no cross-reactivity, antibody...within seconds. A CRISPR-based lineage tracing tool captures rare clones that represent 0.001% of the population...found a way to use the accumulation of indels to measure time. These plasmids with different promoters driving...anti-CRISPRs were identified that inhibit the S. aureus Cas9, a Cas protein that is smaller and easier ...

  39. Light Sheet Fluorescence Microscopy

    Type
    Blog Post
    ...enabling microscopes to visualize the cellular architecture of whole tissues in 3D with unprecedented detail...thin sheet to illuminate a fluorescent sample and captured the reflected light using a different objective...Ultramicroscope was developed using a stereomicroscope to capture large field-of-views (6). For more comprehensive... on several other LSFM configurations’ various features see (4,7).   Light sheet fluorescence microscopy...methods will greatly aid our understanding of the structure-function relationship of the central nervous system...Jährling, C.P. Mauch,  K. Deininger, et al. (2007). Nature Methods, 4, 331–336. Pubmed. P. Keller, M.B. Ahrens...Neuron, 85, 462-483. Pubmed. E.H.K. Stelzer. (2015). Nature Methods, 12, 23–26. Pubmed. M. Weber, M. Mickoleit...

  40. Antibodies 101: Validation

    Type
    Blog Post
    ...your experiments. Capture MS Strategies This approach uses immunoprecipitation to capture proteins from a...been planning, it is a good idea to pause and make sure that your antibody has been appropriately validated...recognition of the target antigen. You want to make sure that your antibody binds to what you expect it to...application and available resources.     Figure 1: Table summarizing the IWGAV five proposed antibody...information for some antibodies, which makes it hard to ensure that you have unique antibodies. An additional ...and then uses mass spectrometry to identify the captured proteins. If the antibody is specific, then you...approach is ideal for validating antibodies for immunocapture applications and is amenable to high-throughput...

  41. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    ...design (meaning the mature processed form of crRNA is fused chimerically to a mature tracrRNA), the spacer... is based on the Cas9 D10A nickase described in Figure 4 of the Cong et al. (2013) Science paper. For ... to select your genomic target, you need to make sure the NGG immediately follows your target on the genome...-allelic cells usually make up the majority in culture unless the targeting efficiency is very high. Q6...NGA', or 'NGC' (if it's within coding region make sure it's a silent mutation). Q8: Is a protospacer ...the Cas9. Again, if it's within coding region make sure it's a silent mutation. Q9: When attempting to use...Restriction Fragment Length Polymorphism (RFLP) (see Figure 4 of the Cong et. al (2013) Science paper). If ...

  42. The Effect of COVID-19 on Liu Lab Plasmid Requests From Addgene Data

    Type
    Blog Post
    ...correspond to publications of popular papers. Figure 1: Plasmid requests by date. Parentheses depict...observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation of the effect of COVID...rough "COVID-19 effect" on order data, it does not capture the fact that different parts of the world encountered...='31668202421', style='text-align: right;' %} Figure 3. An animated map showing the plasmid orders and...during weekends and significant holidays. Figure 4: Plasmid request trajectories suggest an exponential...data likelihood with multivariate Gauss-Hermite quadrature, since the analytical expression for the data...winter break, and COVID-19 on plasmid requests Figure 5: Inferred effects. Values are the inferred percentage...

  43. Droplet Digital PCR for AAV Quantitation

    Type
    Blog Post
    ... the fluorescence amplitude measured for each droplet in each well (Figure 2). A clean ddPCR should have...rate. A rate of 2C/s is recommended to ensure an even temperature change among all of the droplets, but...what if we could repeat this assay 10,000 times to ensure that our titers were as accurate as possible? This...digital PCR involves partitioning a PCR reaction mixture into approximately 20,000 droplets using water-...containing thousands of droplets) can be sufficient to capture the information needed for your PCR experiment....droplet oil that you use should come from the same manufacturer. Otherwise you will end up with poor droplet...reader extracts the droplets from the plate and measures the fluorescence amplitude of each one. Droplets...

  44. Plasmids 101: Colony PCR

    Type
    Blog Post
    ...plate to start liquid cultures. Lastly, you can start small overnight liquid cultures with the clones you...use depends on your preferences. Either way, make sure to test your colony PCR primers before using them...will need to screen.   Saving clones for later culture: At this point, you will want to hang onto your...bacteria-water suspension and use them to start cultures of your positive clones. If you want to store ...ones. Regardless of which method you choose, make sure to use the appropriate antibiotic for selection.... on an agarose gel to determine their size. Make sure to use an appropriate molecular weight standard ...samples before pipetting them onto the gel. The figure above summarizes generalized expected results for...

  45. Cpf1: A New Tool for CRISPR Genome Editing

    Type
    Blog Post
    ... Figure 1: Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Figure from Zetsche...is also much simpler in structure than Cas9’s; only a short stem-loop structure in the direct repeat region...RNAs important for Cpf1 function, they found that mature crRNAs for Cpf1-mediated cleavage are 42-44 nucleotides.... With this information, they turned to a cell culture system to see if any Cpf1 nucleases would exhibit... its proximity to the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively...deletions at the 3’ end of the target sequence. Figure 2: Effects of (A) double and (B) single base pair...Mismatch positions are indicated below each graph. Figure from Kleinstiver et al., 2016. Cpf1’s strength...

  46. Cancer and the Immune System: Deciphering the Relationship

    Type
    Blog Post
    ... way to grow haywire, access and create more vasculature to feed themselves, use the blood stream to commute..., introduced the “Three Es of Immunoediting” nomenclature. 1. Eliminate - Latent tumors are recognized...the host immune mechanisms altogether and escape future immune surveillance. Dormancy of certain cancers... system to the presence of cancer are unable to mature into effector cells in the tumor microenvironment... look at a variety of up and coming therapies. Future directions and open questions in cancer treatment...constantly in flux and is dependent on the selective pressures placed upon it by its environment and the body...immune privilege and predicts reduced survival." Nature medicine 10.9 (2004): 942-949. PubMed PMID: 15322536...

  47. Plasmids 101: Broad Host Range Plasmids

    Type
    Blog Post
    ...identify a few of the features that confer broad host range in plasmids in nature, and provide tips on ... identified some of the features that confer broad host range. These features are: The plasmid origin...characteristics of natural plasmids and how they function in nature can inform us on how to create better recombinant...on how we can harness those features to create broad host range recombinant vectors.  A plasmid’s host...are versatile and adaptable. Variation in ori structure allows plasmids to utilize diverse host replication...and reduced metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid...multiple oris that vary in DnaA-box and iteron structure, host-independent   Building broad host ...

  48. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

    Type
    Blog Post
    ...is to ensure the propagation of themselves and their associated genes. These systems better ensure that...that plasmids are passed down through generations (Figure 1). These so called “plasmid addiction systems”...the mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked by two direct... repeats. In cells where recombination has not occured, methanol induces the production of the toxic MazF...the mok SD resulting in a double stranded RNA structure that is recognized and cleaved by RNaseIII resulting...bioluminescence reporter in vivo in the absence of selective pressure and found that plasmids were maintained at 100%...with plasmid addiction systems While selfish in nature, these systems have been adapted by researchers...

  49. New Tools Enable CRISPRa for Neuroscience Applications

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    Blog Post
    ...sites targeted by specific sgRNAs. Its RNA-guided nature makes CRISPRa much simpler to implement in genome-wide...transcriptional activator to each genome-bound dCas9 (see Figure 1a for the transgenic construct). More importantly...AAV or by mating with Cre-expressing mice. Figure 1: (A) Schematic of the Cre-dependent SPH construct...probe gene-function relationships in defined substructures of the CNS as well as individual neuronal populations...neurons) and Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can be found here. Neuron-optimized...) with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church...including ubiquitous ones such as EF-1α and CAG. Figure 2: Dual lentivirus system with ubiquitous expression...

  50. Tips for Improving Your Next Manuscript

    Type
    Blog Post
    ... quality figure, produce the figure, its legend, and write a results section for that figure. Writing ...next manuscript 1. Be consistent with your figures The figures of a manuscript are the result of a lot of... to my PI about becoming a better writer, I was unsure if I should possibly take a composition class in... individual sections of a paper, the abstract, figures, introduction, etc. so as to thoroughly explain...quilt with your lab mates, you add the individual figures and results to make the final product with an interesting...Scientific Manuscript Writing Scientific Manuscripts: Literature Searching, Reading, & Organizing Write for the...
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