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We narrowed to 1,009 results for: PLE

Showing: 261 - 280 of 1009 results

  1. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    ... is completely degraded. Cloning Grade DNA (high quality/low quantity) – You can usually complete a cloning...transfection-grade While we’ve included a sample of some commonly used kits, please note that Table 3 is certainly...plasmid DNA itself, which can happen if you leave the sample in a lysis buffer for too long or if you get a ...easier than others to prevent quality issues. For example, some kits reduce the risk of ethanol carry-over...you mix specially coated magnetic beads into your sample and use magnets to isolate the beads for the wash...Take your average column-based miniprep kit for example. These kits are designed to handle ~1–5 mL of liquid...can affect the lysis buffer ratio and result in incomplete lysis. You may be able to scale the amount of...

  2. Overcoming the Challenges of Lentiviral Production

    Type
    Blog Post
    ...dish can be easily applied to multiple dishes, remember that handling multiple dishes will take longer and...packaging systems divide the viral elements among multiple plasmids. Second generation packaging systems ...transfer gene. By dividing the viral elements across multiple plasmids, the risk of recombination and consequently...different generations of lentiviral packaging systems please see Addgene’s Lentiviral Guide. While the third...plasmid are not always the best for others. For example, in our hands we find that a double harvest approach...larger genes such as Cas9. See figure 1 for an example of how changing transfection ratios can affect ... volume than with the unconcentrated prep. For example, at Addgene we typically concentrate 100-fold meaning...

  3. Split-BioID: An Improved Method for Studying Protein-Protein Interactions

    Type
    Blog Post
    ...biomolecules. They can be part of multiple dynamic and overlapping complexes that have distinct functions....biotin-containing media. FRET is another example of a protein fragmentation complementation assay that can be used to...proteins that interact with a particular protein  complex as opposed to an individual protein. The Bethune... when the bait is part of a particular protein complex. Not performed in a native cell context. For AP-MS...experiment is called a protein fragmentation complementation assay (PCA). In the Split-BioID method, each...Protein Interactions. Split-BioID is a protein complementation assay where BirA*, an E. coli biotin ligase...proteins Y and Z interact, the two BirA* fragments complement each other to form a functional BirA* enzyme....

  4. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...plasmid deposit process can be broken down into four simple steps: Send plasmid information to Addgene Material... explain how to use our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — ... “Download Deposit Spreadsheet” (see Figure 1). Please read our “How to Deposit Your Plasmids with Addgene... a collection that is greater than 75 plasmids, please contact us at [email protected]. These deposits...any functional consequences of that change. For example, “Mutated aspartate 123 to Lysine (D123K), lowers...indicate type in “Vector Type Other” column. For example, you may enter “donor template vector”. Cloning...37 °C, or room temperature. Growth Strain Please indicate which growth strain Addgene should propagate...

  5. Antibodies 101: Stripping and Reprobing Western Blots

    Type
    Blog Post
    ...membrane will remove some of your sample —and harsher stripping means more sample loss. If you are still optimizing...you want to detect more than one protein in your sample? Or what if something weird happened during your..., almost like new. This approach preserves your sample and saves you from having to run a whole new gel... of time that you want to probe your blot with multiple antibodies. You’ll want to set up your blot with...stripping solution (see below) to minimize loss of your sample from the membrane. Next, plan out the order of ...stripping methods. If you are planning to detect multiple proteins through several rounds of stripping and... the weaker binding ones first to preserve your sample. Pro tip! Because some protein is lost each time...

  6. AAV Vector Quality Control: Going the Extra Mile with NGS

    Type
    Blog Post
    ... DNA and processed the mixed sample in our pipeline (see Figure 2, sample 2). When we blindly analyzed...questions are especially important when producing multiple vectors in parallel because the risk of accidental...in-house sequencing capabilities and Seqwell’s plexWell technology, we can now perform NGS at Addgene ...these known impurities, as was the case for clean sample AAV-44362. In addition to these expected hits, ...to “random” genes. Do these hits always mean the sample is contaminated? No - what really matters is the...of hits to the same sequence (gene). In a clean sample, there are typically fewer than 10 hits to a given...>100 hits - immediately raises a red flag. For example, AAV-68544 contained 220 hits to the CHRM4 gene...

  7. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

    Type
    Blog Post
    ...with increasingly complex experimental conditions. The ability to construct more complex, multi-fragment...fragments in a single “one-pot” reaction to form complex, multi-insert modular assemblies that enable biosynthetic...enzyme cutting results in 5’ four base overhangs complementary to the overhangs of the assembled inserts. After..., the BsaI site is no longer present, allowing multiple fragments to be assembled in an ordered, pre-determined...assembly Intermediate 5- or 12-fragment assembly More complex 24-fragment assembly Single insert cloning efficiencies...Sequencing of blue colonies showed the expected complete sequence for the lacI/lacZ genes (1), while sequencing...single omission should block the formation of a complete assembly that would result in a blue phenotype...

  8. Experimenting with New Careers while in Grad School

    Type
    Blog Post
    ...academia One recent example: Don't call me a dropout: Why science needs more people to quit the lab. ...re provided throughout your PhD program.   Find people who will support your professional development ...are not as supportive of your career goals, find people who are. I wanted to do fun science activities ...activities with the public, so I reached out to people who were educating the public and organizing events. I contacted...attention on social media. I gained a concrete example to use to promote future attempts and learned a...try new things, develop new skills, and meet new people. There were visiting seminar speakers each week...meet with various professionals, and there were multiple avenues for professional development including...

  9. Drew Endy Introduces the Biobrick Public Agreement Plasmid Collection

    Type
    Blog Post
    ... useful they are implemented in most materials that have been engineered. For example, Boole’s logic shows...biological engineering openly so as to benefit all people and the planet. For his many efforts in open science...BioBricks Foundation to distribute the physical DNA samples, for when that's the preferred mode of receiving...phrase we like to use is 'the parts must flow.' People must be able to use genetically encoded functions... for engineering an entire cell. Typically when people use plasmids, they are trying to do one thing at...Integrase Logic (BIL) gates collection of parts implements the basic logical functions, which we know and..., computers, and now through BIL gates we've implemented them in DNA. We thought these were so much fun...

  10. Cre-ating New Methods for Site-specific Recombination in Drosophila

    Type
    Blog Post
    ...to make complex genome modifications in Drosophila using multiple recombinases. To make multiple, precise...recombination by KD, B2 and B3 resulted in complete or near-complete excision of the stop sequence. In contrast...to DNA damage and developmental aberrations. In multiple systems, Cre itself, without the presence of a...toxicity, but with the caveat of partial rather than complete recombination. If you’re looking to use site-specific... high recombinase levels. These data show that multiple recombinases can be used together to designate...for specific recombination patterns that require multiple recombinases. These tools should further enhance...editing capabilities in Drosophila. References: Multiple new site-specific recombinases for use in manipulating...

  11. When Fidelity Matters: A frank discussion about ligase fidelity

    Type
    Blog Post
    ... (5,12,13). For example, in the Ligase Detection Reaction (LDR), probes complementary to the sequence ... extended to multiplexed probe sets that allow the simultaneous interrogation of multiple potential SNP...ligation of fully base-paired nicks with no gaps. For example, DNA assembly methods, such as Gibson Assembly®...without the exo/polymerase generation of annealed complementary regions. Why use a high fidelity ligase? Numerous...extends earlier studies through a high-sensitivity multiplexed format (21, 27). This methodology has been used...separate pools were prepared, each with a different complement strand covering all 16 possible NN pairs in the...Stefan V., and Göran Magnusson. "Sealing of gaps in duplex DNA by T4 DNA ligase." Nucleic acids research 10.5...

  12. Plasmids 101: Codon usage bias

    Type
    Blog Post
    ... encode which amino acids (see example to the right). With such simple rules, you might think it’s easy...codons unaccounted for. The result is redundancy; multiple codons encode single amino acids. Evolutionary...varied codon preferences among organisms aren’t completely understood, but some possible reasons include...resources for the entire translation process. For example, during high growth rate conditions, E. coli preferentially...encoded by highly abundant, charged tRNAs. For example Tuller et al. found that translation efficiency...translated regions a chance to fold properly. For example Pechmann and Frydman found that tracts of non-optimal...producing and charging specific tRNA pools. For example, tRNAs used in genes encoding amino acid biosynthetic...

  13. Important Considerations in Optogenetics Behavioral Experiments

    Type
    Blog Post
    ...fully summarized in a short blog post, but some examples I’m personally interested in include: intracranial... is an approximation of reward or the sense of pleasure that the animal perceives through taking a drug... also models reinforcement but is considerably simpler than ICSS. The test chamber is divided into two...expression of the early response gene c-fos. For example, using Chr2 stimulation should result in robust...c-fos expression to get the evidence you need. For example, as shown in this study [3], hippocampal neurons...mouse is placed in a novel context. As a proof-of-principle test, the authors were able to show that HR blocked...involved in regulating the behavioral output. For example, if you are interested in studying a brain region...

  14. The Strength of Story Telling

    Type
    Blog Post
    ...on living as a scientist in Beijing, talking to people about taking beautiful photographs of their studies...Story Telling at work Don’t believe me? Here’s an example of storytelling to answer an informational interview...it worked out for all of us.” This is a classic example of the STAR (Situation, Task, Action, Result) interview...than I’m able to produce. But it’s also a great example of storytelling – you get context, time, cause,... and wide, keep your website up to date, invite people to ask questions, be approachable. Engage with ...technique you have in your arsenal to make sure people pay attention. Highlight your lowest points and...into a scientific story, but it will ensure that people pay attention. Practice Nobody gets good overnight...

  15. Antibodies 101: Chimeric Antibodies

    Type
    Blog Post
    ...will not affect single-plexed experiments, it needs to be addressed when multiplexing. It is imperative that...cannot be indirectly probed simultaneously on the sample. What to do?  If you’ve ever found yourself facing...the constant regions from an alternative isotype (purple, mouse). Depending on the cloning process, all ...conversion, gives users the flexibility needed for multiplexing. In the case above, you can’t costain with your...visualize both targets in parallel (Fig. 2). For example, if both of your antibodies are rat IgG1, then ...antibodies with the same isotype can not be used for multiplexed indirect staining methods since the secondary...the new Fc region. In addition to increasing multiplexing capabilities this process is frequently used...

  16. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

    Type
    Blog Post
    .... HDR is crucial for certain applications, for example, correcting the allele that causes sickle cell ...decision. Our conclusions reinforce a key scientific principle: that understanding a biological process in detail...manipulate that process. We began our research with a simple question: how do Cas9 and sgRNA interact with and...tightly to three of the four strands of the cleaved duplex, while the fourth strand (the released strand in... in Figure 1 below) was free to anneal to complementary ssDNA molecules in vitro. Figure 1: Cas9...Cas9 binding to the cleaved DNA duplex. Image from Richardson et al., 2016. We next wondered if we ...discovered that single stranded donor DNA: Complementary to the released strand, 127bp in length, With...

  17. Thoughts on the Future of Research 2015 Boston Symposium

    Type
    Blog Post
    ...that need people who are data oriented, scientifically minded, and able to communicate complex information...research and potential solutions During the first couple of sessions of the symposium it was clear that,...postdocs. Unfortunately, even when universities have simple postdoc numbers sorted out there can be gaps in...Postdoc: An Institutional Perspective. This lack of complete information does not, however, prevent us from...these positions are available and that many of the people who work in them are incredibly happy with their...need to be able to network and interact more with people who have taken a variety of career paths and should...collaborators, others you know in the field, and even people who aren’t in the field. One PI on the panel even...

  18. Behind the scenes: Addgene’s new search engine and more

    Type
    Blog Post
    ...remedy this, throughout the year we rolled out multiple changes to our website, including the recent homepage... new website search. Tackling findability from multiple angles    We assembled a cross-team team, with...interface.   The team approached the issue from multiple angles: the overall user experience of our website...website navigation paths, the organization and completeness of our information, and the accuracy and relevance...relevant to a specific query and category. For example, a search scoped to plasmids shows filters for ...appear in our data into a single filter value. For example, there is only one viral prep filter for promoter..., human synapsin, hSynap. Matching We capture multiple variations of the same information in our search...

  19. dTAG - You're it!

    Type
    Blog Post
    ...degradation with dTAG-13. Example experiments See our recent papers noted below for examples of dose-responses... pharmacological inhibitors including rapidly depleting a protein-of-interest, increasing target selectivity...FKBP12F36V-tagged proteins. The dTAG molecule induces ternary complex formation between the FKBP12F36V-fusion and the...happy to share aliquots of dTAG-13 in the meantime, please address your requests for dTAG molecules and any...parental cells (or cells expressing a control -- for example, an FKBP12F36V-tagged LACZ) with dTAG molecules...5 in Nabet et al., Nature Chemical Biology for examples). Monitor neo-substrates Small molecule degraders...neo-substrates varies in different contexts (for example, IZKF1 and IZKF3 are cell-type specific transcription...

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

    Type
    Blog Post
    ...resources of a cell. The objective is to give people the complete toolbox for taking on very large genetic ... do as genetic circuits become larger and more complex? Voigt: If you look at all the products of biology... on genes at different times. It requires more complex synthetic networks. Addgene: What hurdles did you...overcome and what hurdles remain? Voigt: There are a couple of hurdles we haven’t yet overcome. For this particular...CRISPR chemistry for a NOR gate. But there are a couple of problems. You couldn’t do it without repeating...first one worked in each case. Sometimes in CRISPR people talk about off-target interactions. That’s relevant.... There haven’t been enough genetic parts for complexity. We have characterized almost 600 terminators...
Showing: 261 - 280 of 1009 results