...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 ...

...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...

...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...

...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...

...potential applications. Challenges of existing mutagenesis methods Several continuous diversification methods... challenging for users to achieve targeted diversification of all nucleotides within a defined region ...Chang Lab have created methods that introduce diversification by targeting a site of interest with dCas9 ...level of variation that can be introduced.   Diversification is limited due to the integration of oligonucleotide...increasing the standard mutation rate seen during DNA replication by 120-fold over background. Due to its modular...only ~2-fold over nCas9 levels. Two sets of modifications were also made to PolI3M to increase the mutation...more than one mutation near the target site. Applications of EvolvR The Dueber Lab used EvolvR to discover...

... 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...

...led to their application for basic and translational research.  Basic research applications of scFvs: ...blood clearance, which is useful for imaging applications; better tissue penetration which is useful for... for therapeutic and imaging applications; and reduced immunogenicity when administered in vivo due to...from blood can be a drawback for therapeutic applications where longer retention times often increase ...since it’s genetically encoded.   Translational applications of scFvs: Just like antibodies, there are many...2019).Many of the scFvs designed for oncology applications are reformatted as bispecific molecules which... autoimmune, and/or inflammatory therapeutic applications. Antibody fragments such as scFvs are also used...

...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...

...pre-treatment levels due to the viral reservoirs located in latently infected cells. Although ART-treated...toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating a true “shock and kill” response. Cutting HIV...create mutations in genes essential to HIV-1 replication. However, this approach could permit some viral...along with Cas9 in the T-cell line 2D10. PCR amplification and Sanger sequencing of pooled samples showed... HIV-1 could escape from CRISPR/Cas9-induced modification targeting either the LTRs or essential genes...genes. Many of these escape mutations were located near the Cas9 cut site, leading Wang et al. to conclude...lessons learned in pursuing one technological application can benefit many others and accelerate the pace...

...ligation using SpyLigase Building off their SpyTag/SpyCatcher system for protein-peptide locking, Mark Howarth... a critical role in development and are often implicated in disease progression. One of the major challenges...challenges in signaling research is the inability to replicate the spatio-temporal precision with which signaling... of complex biological systems requires both a catalog of molecular players and contextualized knowledge...the most abundant forms of post-translational modifications in cells and research into its many roles in...Alex Schier's lab, designed for expression and purification of Cas9 protein in Rosetta E. coli. New Lentiviral...FokI-dCas9 expresses Fok1 nuclease domain fused to catalytically inactive Cas9 DNA-binding domain in mammalian...

...the optimal location. It is also important to have good information on the exact location of the TSS. ...appreciable levels of modification. Whole-genome sequencing of cells modified by CRISPR indicates that the consequences...specific base edit, or modulation of gene expression. Location and sequence are important considerations for ... gRNAs. For indels, it's not so important what location in the gene you target, but it is important that...fewer sequences to choose from). Finally, for HDR, location is much more important because you have to target...target site is far more constrained by the desired location of edit; efficiency decreases dramatically when...alternative to HDR for introducing edits. The same locational constraints are even more exquisite for the so-called...

...species can be indicated in the “Species Other” column. Relevant Mutations Indicate the amino acid...usually very easy — our tech transfer team will communicate with your organization’s technology transfer ...name and match the name as it appears in its publication or as it is commonly known in your lab. Plasmid...descriptive purpose focuses on the practical application of the plasmid and will help a scientist outside...reagent/antibody, unspecified, or other. If other, indicate type in “Vector Type Other” column. For example...or room temperature. Growth Strain Please indicate which growth strain Addgene should propagate your...For plasmids that contain the R6Kγ origin of replication, choose the Pir1 strain. If your plasmid cannot...

...able to replicate, and for a virus to cross a synapse from one cell to another, it must replicate. Modified...like how our stem cells are housed in specific locations and give rise to progenitors during growth and...infects one cell (the “starter cell”). It then replicates within that cell, and nascent RABV cross synapses...infection occurs, subsequent cycles of viral replication and retrograde transmission occur, spreading ...limitations. First, these high levels of viral replication are toxic. Second, infecting subsequently higher...spread monosynaptically from these cells. These modifications take advantage of the fact that RABV is an enveloped...G-deleted RABV will infect the starter cell. RABV replication will then occur and the G protein (supplied in...

...your experiment (all well validated for your application, of course!) When the stars are aligned and the...factors that affect isotype choice. Target and application should be considered The first question you may... a strong signal and to work in a variety of applications. The desired specificity of your secondary antibody...affect my experiment?” Well, it depends on the application and target. If you are running a basic western...target is expressed at very low levels or your application requires a high degree of affinity, then species...considered when choosing an antibody. In some applications probing species-on-species, i.e. using a mouse...mouse IgG2a and rat IgG1 antibodies in the same application, using a Goat anti-mouse IgG2a cross-adsorbed...