We narrowed to 1,017 results for: PLE

...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...

...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...

... 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...

...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...

... 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...

...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...

... (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...

...for multiplexed targeting with Zhang lab plasmids? A6: For multiplexing CRISPR to target multiple genome...4 of the Cong et al. (2013) Science paper. For example, if you want to use double nickase, you could express...want to use wildtype cas9 or double nickase). For example, if you want to target two genomic loci with wildtype... vector, for one well in 24-well plate. A good example/reference is Ran et al. (2013). In the case where...putting in the full target site in the donor. For example, choosing target sites that span the knock-in gene...selection marker to select transfected cells (for example, plasmid with a puromycin resistance cassette, ...individual genotyping (using Sanger sequencing, for example) on each colony in order to screen for positive...

... 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...

...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...

...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...

...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...

.... 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...

...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...

...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...

...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...

...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...

...reducing editing from ~30% in control samples to 0-10% in Acr samples. Acr proteins can also be used to prevent...assembly of the crRNA-Cas complex (Zhu et al., 2019). Inhibiting DNA cleavage Example: AcrIIC1 binds to the...protein while others can inhibit CRISPR enzymes from multiple species of bacteria.  A note on anti-CRISPR nomenclature... of discovery (Bondy-Denomy et al., 2018). For example, AcrIIA4 mentioned above indicates that it’s the...activity one of three ways: Inhibit DNA binding Example:  AcrIIA4 blocks Cas9’s interaction with the PAM...site (Harrington et al., 2017). Inhibit crRNA loadingExample: AcrIIC2 interactions with Cas9 interferes ...and spatial control of CRISPR-Cas activity. For example, fusing AcrA4II to the photo-sensitive LOV2 domain...

...Plasmid Kit Multiple Lentiviral Expression System Kit (Kit #1000000060) The MuLE (Multiple Lentiviral ...several different viral vectors to choose from, for example gamma-retrovirus, lentivirus, Adenovirus, and Adeno-associated...and lentiviral envelope plasmids are listed here. Please note that both lentiviruses and gamma-retroviruses...combined with high-throughput sequencing to perform multiplexed analysis of cells pre-treated with a compound...Transfer Vectors    Expressing your gene of interest pLenti-puro (Plasmid #39481) You can clone your gene ...We are constantly improving our CRISPR reagents. Please check www.genome-engineering.org for the most up-to-date...system facilitates the simultaneous introduction of multiple genetic alterations into mammalian cells. The ...

...predecessors. Changes were implemented and I had to adapt accordingly. For example, the quarterly and annual...annual NPA meeting to learn more. Having met many people at that conference, I later became involved with...volunteered for various tasks as things came up. For example, I updated some sections of the NPA’s website. ...volunteering, you can gain experience working with other people mostly online and on monthly phone calls. This ...distance communication and learning to work with people despite the distance improved my verbal and written...understand how to work with many different types of people both above and below you. During my time at the...NPA. You must accommodate many different types of people in order to harness their strengths and work effectively...