...additional origins of replication compared to monomers, they are also able to replicate at a higher frequency...expected fragments for that plasmid. This phenomenon indicates the presence of a multimer.   Fig. 1:...the right, with the entire monomeric sequence duplicated in tandem, resulting in a plasmid that is twice...observed to exist in various multimeric forms (e.g. concatemers, dimers, trimers, tetramers). Since then, multimerization...other methods below should help in multimer identification.   Fig. 2: Gel image of Plasmid 61564...within a plasmid and, therefore, allow for the identification of plasmid multimers. Capillary Gel Electrophoresis...multimerization. One study by Bacolla et al. (2011) indicated that strain HB101 produced the highest yield of...

...someone has replicated it precisely. You can find a great discussion of different types of replication in Nosek...perfect story for a “high impact” publication can lead to the publication of rare flukes showing positive...that fall into this category include performing experiments with too few replicates leading to suspect ...these positive results will be published. The publication record will then show that the tested hypothesis...reproducible publishing practices. One example is the publication of peer reviews so we can see how and why papers... also means that you’re more likely to get a publication (for better or worse, the currency for success...review of experimental design as part of the publication process (Nosek et al 2018). In this ideal situation...

... gene’s transcription. Sometimes enhancers are located thousands of base pairs away from the gene they...created a dCas9-based CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) method. The ...could only detect chromatin interactions at one location in the genome at a time (Liu et al., 2017). Recently...at once (Liu et al., 2020). CRISPR Affinity Purification in situ of Regulatory Elements: The original...biotin ligase a gRNA(s) targeting a single genomic location of interest  When these three components are ...together in terms of where they are physically located. DNA is then sheared into pieces small enough to... original CAPTURE and CAPTURE 2.0 methods.   Applications of CAPTURE 2.0 CAPTURE 2.0’s increased rate ...

... tools for familiar applications. But how does one invent a tool for applications that don’t even exist...phenomenon, improve the tool, and develop new applications for magnetoresponsive proteins. Motivated by...organisms) are opaque, which makes most biomedical applications of these tools difficult or impossible. But ...tool with magnets would enable new research and applications that don’t yet exist, but it wasn’t clear that...changes in response to a ~25 mT magnet. Gray regions indicate times when the magnetic field is on. Image reproduced..., in response to a ~10 mT magnet. Gray regions indicate times when the magnetic field is on. Image reproduced... shows a wealth of potential, and while many applications are still yet to be demonstrated (or invented...

...lentivirus, AAV does not possess the ability to replicate itself and depends on helper viruses like adenovirus...herpes simplex virus to facilitate efficient replication. The most common production system, however, ...RNA genes; and the pRepCap plasmid containing replication factors and serotype-specific capsid genes.  ... steps of AAV production, iodixanol gradient purification, and AAV titration by qPCR.   Pro tip! An optional...al., 2010). For certain in vitro and in vivo applications, you can consider using unpurified AAV from ...Benyamini et al., 2023) to save time and cost.  Purification To purify your rAAV from your supernatant, you...recommend using a Tris-based lysis buffer and sonication.  The obtained virions from cells and supernatant...

... to be crazy complicated! Listen to the full podcast for more great science communication tips or listen...tons of ways you can get involved in science communication. In this second half of our conversation with...many things you can do start your own science communication student group and get more involved with your...is a graduate student group with the goal of communicating science to the general public and training the... the next generation of science communicators. In this chapter we learn about some of their events - science... peer-to-peer feedback in effective science communication. Chapter 7: The Goal of the SITN Podcast - Vini...Pint" event is the best way to get a Science Communication group started. It's as simple as getting people...

...my opinion, it was more important to communicate the applications of CRISPR over the details of how it...m a father of three, a scientist and science communicator, and I strive to be a volunteer and contribute...recent experiences. STEM in elementary schools - catching them early I’m fortunate to live in a town with...unlike elementary schools, middle schools have dedicated science teachers. I tried contacting our school...simplistic and I would insult the students. Too complicated and I would quickly lose their interest. I won...great feedback from teachers and other science communicators.  What can CRISPR do? I spent more time on...children and you want to make sure their STEM education is supplemented. A 2009 study by the NSF showed...

...valuable to scientists, there are some potential complications that must be considered when using this method...alternative approach, which avoids many of these complications, is to directly deliver a ribonucleoprotein ...used for most of the current genome engineering applications of CRISPR including: generating single or multi-gene...pathways. This makes Cas9 RNPs useful for CRISPR applications where limited expression of Cas9 is required...development. David Liu’s Lab has demonstrated the use of cationic lipid-mediated methods to deliver Cas9-gRNA RNPs... of eventually using this method for in vivo applications. This technique utilizes Cas9 proteins harboring...CRISPR experiments can reduce many potential complications, there are additional factors to take into account...

...If you use a kit for DNA purification or if you use a DIY purification protocol, you might have noticed... mutations in single stranded DNA undergoing replication.   DNase on the other hand can digest and degrade...degradation during plasmid DNA extraction and purification from E. coli is the presence of the protein ...most common E. coli strains used for plasmid amplification are endA negative, meaning the gene has been...chelating agents that can be introduced in the purification protocol to remove endonucleases activity. For...controls the pH, while the EDTA chelates any divalent cations like Mg2+, preventing the activity of DNase. While...do with these samples. This is because divalent cations are essential cofactors for many tools used in ...

...logistical barriers, allowing people to access a wide catalog of plasmids from all over the world. This idea,...into the accessible. Physical access Addgene is located in the USA, but our materials are available to ... items to countries with a distributor. Arrows indicate the year we entered into an agreement with a distributor...accessibility to researchers around the globe. Addgene’s educational resources As Addgene built out our scientific...guides, protocols, and collections of 2023. Educational Resource Top Five by Views Guides CRISPR...Systems AAV Plasmids Blogs Origin of Replication What is a Plasmid? How to Design Your gRNA for...countries. In Table 1, you can see our most popular educational resources for each type of resource we offer....

...the other to guess a category by listing off things that fall into that category? Okay, let’s play. I’...’ll list the examples and you try to guess the category: ELISA...qPCR...Digital droplet PCR...DNA dot ...concentration of viral genomes (by qPCR or other DNA quantification methods - see below), since each viral particle...value, physical titers don’t necessarily give any indication of the infectious titer. The infectious titer...affected by many experimental factors. To list some complicating factors for quantifying AAV: qPCR efficiency...optimized our assay in a few ways:  Absolute quantification by qPCR requires generating a standard curve...plasmid standards. We validate our absolute quantification by using the universal AAV Reference Standard...

...advancements. Consultants can help these teams develop communication strategies, or understand, and publish on the...success. Or the client might need help with the intricate task of designing a strategic vision for a portfolio...investments over the next 3, 5, or 10 years. Complicating these challenges is the fact that they occur...chemotherapies, or developing innovative mobile phone applications and services that help diabetic patients manage...knowledge from previous consulting experiences, educational training, and conferences, and synthesizing this...that gives the client a defined path forward. Communication is key as ideas must be relayed quickly both...clients and experts. Hence, active honing of communication skills is a must to be successful in this field...

...cellular impediments. FRET has been used in many applications, including drug discovery by big pharma (i.e... acceptor doesn’t need to go to the same exact location as the donor. As long as some of the acceptor ...C-terminus of your target protein, but even these modifications can affect the structure or function of certain... close to the donor, faulty delivery to other locations in the cell are not a problem, as long as they...The poly-histidine tag often used for protein purification is a reliable FRET acceptor labeling site when... way as NTA-sepharose beads used for protein purification: the NTA binds to the His tag on your protein... used to fluorescently label antibodies, and purification is a snap using thin layer chromatography (Fessenden...

...overall. Communication is the issue Much of the problem can be attributed to communication, and in particular...particular communication about experimental methods and approaches. It’s very easy to think that the methods...that they were doing something differently. Communication comes in many forms—from transparency about ...them troubleshoot and understand. All of this communication and sharing fits together to make for a transparent...for pushing for more transparency in science communication falls on the shoulders of journals and publishers...can feel like yet another hurdle in front of publication when all they really want to do is get the paper...we take our responsibility to push for clear communication of science very seriously. That’s why at Cell...

...plasmids and their potential educational uses. We believe that biology education is something that should ... expensive tiny amounts of reagents are, how complicated (and again, expensive) the different machines...desire to create a set of low-cost, easy-to-use educational biology activities – that any school could afford...proteins without the need to handle a living, replicating organism. Furthermore, cell free systems can ...in adapting your research ideas into hands-on educational activities using the BioBits platform, please...BioBits™ Explorer: A modular synthetic biology education kit." Science Advances 4.8 (2018): eaat5105. PubMed...BioBits™ Bright: A fluorescent synthetic biology education kit." Science Advances 4.8 (2018): eaat5107. PubMed...

... a bacterial origin of replication (ORI) will not allow for plasmid replication in mammalian cells, for... their bacterial counterparts, including how replication occurs and whether selection is necessary for...the nuclear genome, in the absence of plasmid replication, the foreign DNA will be degraded or diluted ...always mission critical to make sure the plasmid replicates in the cell, as many of these experiments are...expressed in trans within the cell for effective replication. Cell lines expressing the Epstein–Barr virus...antigen (293E or 293T cells), allow for episomal amplification of plasmids containing the viral EBV or SV40...contain an ORI since the integrated DNA will be replicated as part of the genome. Because the foreign DNA...

...check its subcellular location and solubility. If it’s membrane-bound or located in a subcellular compartment...nuclear- or DNA-binding proteins, you may need to sonicate your lysates in order to release the proteins ...will simply load an extra ladder or technical replicate (ie, leftover sample) to fill the relevant wells...membrane into the psi membrane, that’s a good indication that the p membrane pores are too large.  Transfer...likely technical. White circles, for example, could indicate that you had air trapped between the membrane ...during the transfer, while dark splotches could indicate dirty equipment, degraded or improperly mixed ...using an antibody validated for your protein and application of interest or validating an antibody yourself...

...recombinase polymerase amplification (RPA) using a commercially available amplification kit. In this case,... RNA is extracted and subject to isothermal amplification by primers that target the E and the N2 gene...reverse transcriptase to produce cDNA. Isothermal amplification is done at one constant temperature, eliminating...rapid detection.  Highlights: Cas13a is not catalytically active in the presence of two or more mismatches...that exhibit fluorescence upon cleavage. Upon amplification by the RPA primers, the Cas12a-crRNA binds to... SARS-CoV-2 detection, AIOD-CRISPR without preamplification was able to detect as low as 1.2 copies DNA..., field-deployable nucleobase detection and identification using FnCas9 (FELUDA) Sensitivity: – 110 femtomolar...

...resistance cassette, and the R6K origin of replication to allow replication only in hosts carrying the bacteriophage...chromosome. These methods roughly fall into two categories: those that rely on exogenous enzymes and those...Lambda Red recombineering, which uses enzymes domesticated from the Lambda bacteriophage, is arguably the...enzymes can also accomplish the desired genomic modification. This technique is frequently called allelic...recipient of interest. Because pDS132 cannot be replicated in the recipient, the only antibiotic resistant...exchange. To do this, we performed the following modifications to pDS132 (Philippe et al., 2004): Replaced...Lazarus et al., 2019). Additionally, to facilitate modification of strains where conjugation may be difficult...

...conflict is not always what was communicated but how it was communicated. The next step is to come up with...of lapsed communication. Making some assumptions about how something should be communicated is necessary...skill in and of itself. Conflict can also be categorized as either “personal” or “substantive”. A personality-based...traced back to one of a few trouble spots:  Communication (or lack thereof): Information sharing gets ...organization gets, and creating new avenues of communication can create as many problems as it can solve....meeting? Different teams frequently prefer to communicate in different ways, so the source of the conflict...with a viable process that will avoid such miscommunications in the future. Assumptions: Making incorrect...