...variety of other undesired outcomes, including one loxP site insertion and one NHEJ-repaired indel, often ...around the desired insertion site to search for potential gRNAs. As with other homology-directed repair experiments...with your desired insertion feature (e.g., a fluorescent protein), you can amplify the desired region using...the power of ssDNA donor molecules for homology directed repair. Using long ssDNA donors, the Gurumurthy...arduous. First, you’d construct a plasmid with your desired changes, long homology arms and a selection marker...pseudopregnant female mice. If you obtained the desired chimeric mice, you would breed them many times,...times, hoping to produce a mouse with the desired edit. You’d also need to remove the selection marker, likely...

...Constitutive but requires T7 RNA polymerase Promoter from T7 bacteriophage Sp6 Constitutive but requires Sp6 RNA...eukaryotes. In bacteria, the core RNA polymerase requires an associated sigma factor for promoter recognition...in eukaryotes is much more complex. Eukaryotes require a minimum of seven transcription factors in order...

...assay run time to two days.  Indirect or direct detection method Indirect detection is the most common... to primary antibodies). Direct westerns are simpler and faster than indirect ones and can be used in ...well, and multiplexing is challenging and limited. Direct westerns typically rely on primary antibodies conjugated...sensitive to lower amounts of protein, and can require more antibody or loading sample, which may not ...conjugate, and changing your reporter molecule requires either re-conjugating or re-purchasing your primary... like the mitochondria or nucleus, will likely require a harsher lysis buffer than proteins easily available... also add time to an already lengthy protocol, require the use of hazardous chemicals, and are not always...

...Generation Slower Faster Cofactors/Substrates Required Not Required Signal Strength Lower Higher Sensitivity...Higher Post-translational Modification Not Required Required Photobleaching/Phototoxicity Not susceptible... North American firefly(Photinus pyralis) 560 61 Luciferin ATP No Japanese firefly(Luciola cruciate...cruciate/lateralis)Italian firefly(Luciola italica)Southern Russian firefly(Luciola mingrelica) 562 64 Luciferin...bioluminescent organism for most people is the firefly (Photinus pyralis) and perhaps not surprisingly... the initial article describing the cloning of firefly luciferase was published in 1985, several studies...since photons are simply measured – they are not required to initiate the reaction. Table 1 compares some...

...experiments that require exogenous protein, successful induction of RNAi only requires the presence of ...as the entire culture can be harvested instead of microscopic isolation. The plasmids required to conduct...tools exist to perturb biological function in a direct or unbiased way at the DNA, RNA, or protein level...level. The “correct” choice of tool requires careful balancing of the inherent advantages and limitations...Figure 1). The plant kingdom has expanded its repertoire of Dicer enzymes and these different Dicer-like... of an RNAi-inducing transgene. Typically, the desired target of RNAi is expressed as inverted repeats...and Stainier, 2020). Additionally, a study that directly compared CRISPR to RNAi in the context of a high...

...three are required for recombineering with a dsDNA substrate; however, only Beta is required when generating...and no longer requires drug selection.  One drawback to this approach is that it requires the generation...based on homologous recombination. It allows for direct modification of DNA within E. coli and is independent...occur in a test tube. The donor DNA substrate only requires ~50 nucleotides of homology to the target site...2) if the dsDNA was short enough, a ssDNA whose entire complementary strand was degraded. Beta: Beta protects...ssDNA target in the cell.  Only Beta expression is required for recombineering with an ssDNA oligo substrate...substrate should be ~70-100 nucleotides long with the desired alteration(s) located in the center of the sequence...

...Buy American, Hire American" Executive Order which sets broad policy intentions directing federal agencies.... or postdoc. Foreign national researchers who require visas, permanent residence or citizenship to continue... likely that an international candidate will be hired for a role that is closely aligned with their education...degree or its equivalent and state licensure, if required to practice in that field. H-1B work-authorization...research organizations, and organizations that require the H-1B employee to work at one of the first three...application meaning that the category does not require a foreign national to have an employer or a job...the individual’s past work on the field. NIW requirements stress “educational background, expertise, publications...

...No Purified Enzymes Required No No Yes Yes Lab Made Competent Cells Required Yes No No No Comparison...Site-directed mutagenesis (SDM) is one of the key tools researchers use to prove causation in molecular...small point mutations, large deletions/insertions require complicated, often costly in vitro assembly methods... Linearized Plasmids After PCR). Current site directed mutagenesis methods Current SDM methods rely on...you design two oligonucleotide primers with the desired mutation, then use those to PCR amplify the template...but these methods can be expensive because they require particular enzymes, some of which are proprietary...above, you design two primers, each containing the desired mutations, to amplify both strands of a plasmid...

...the entire axial volume of a sample. Therefore, when a large number of optical slices are acquired all...This post was contributed by Doug Richardson, Director of the Harvard Center for Biological Imaging and...the microscope objective evenly illuminating the entire (wide) field of view. Fluorescent light traveling...visualization. Illuminating a sample in the opposite direction to fluorescence collection is referred to as “...increases the speed at which a confocal image can be acquired. Assembling an image pixel by pixel is slow; therefore...hole is illuminated at a time, the image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy...limiting photobleaching. Similar to widefield, the entire field of view is excited at one time and captured...

...table below) have different PAM requirements. These different PAM requirements allow researchers to target...encodes a single crRNA. The direct repeat regions contain sequences required for processing pre-crRNA into...variants are being discovered all the time and the requirements of these different systems can vary (for example...processing foreign DNA as well as the RNA guides required for endonuclease function, when used for genome...genome engineering, the only CRISPR protein required is the Cas9 endonuclease or a variant thereof. This ...contains both the 20 nucleotide target sequence to direct Cas9 to a specific genomic locus and the scaffolding...researchers simply need to express a gRNA designed to direct Cas9 to their target sequence of choice (see tips...

...Cas9s with looser PAM requirements compared to SpCas9. xCas9 has a PAM requirement of NG, GAA, or GAT, ...A-to-G.) In short, the impaired Cas9 localizes the deaminase to where the edit is desired, and it performs ...with a variety of impaired Cas9 proteins (such as SpRY) with altered PAM requirements, giving it more flexibility... al., 2019), it does not require an expertly positioned PAM without undesired bases nearby that may undergo...Site-specific deletions and knockouts Some experiments may require the introduction of a mutation at a specific site...alternative PAM Cas9s can be found in “The PAM Requirement and Expanding CRISPR Beyond SpCas9.” Single base...flexibility. A consideration with this tool is that the desired edit will be performed on all relevant bases within...

...: dCas9-targeted point mutations for directed evolution Directed evolution is an important method for ... function, strength, and binding (e.g. DHS-seq, FAIRE-seq, and CHIP-seq), genome-wide identification and...pooled libraries enable researchers to screen the entire genome for genes that regulate a wide variety of...the NCC-1 library of approved drugs against the entire kit; a parallel analysis that successfully identified...Researchers can use plasmids from the PRESTO-TANGO kit directly for drug screening or easily shuttle the optimized...optimized, expression validated GPCR sequences to any desired backbone.  Kroeze, et al. Nat Struct Mol Biol....oligonucleotides (oligos), and thereby rapidly create desired allele combinations and combinatorial genomic libraries...

...familiar with when talking about IF, direct and indirect. For direct IF, researchers use a single antibody...allowing one to “directly” visualize their target. In contrast, indirect IF requires two antibodies: a...post to learn more about these techniques!  Direct vs. indirect immunofluorescence There are a couple different...Figure 1: Comparison between how direct immunofluorescence and indirect immunofluorescence work. Image ...with BioRender.com   Indirect IF is a much more common approach than direct IF, so we will focus on... a primary and secondary. For indirect IF, the primary antibody targets the antigen of interest but is...on this technique here. One of the reasons that indirect IF is more common is that secondary antibodies...

...flow cytometry can use both direct and indirect staining methods. In direct staining, the primary antibody...binds to the primary antibody. Direct staining is quicker than indirect staining and eliminates potential...assessed for the desired parameter. A droplet containing a cell that is positive for a desired parameter is...with protocols that require fixation and permeabilization as these methods require damaging the cell membrane...as it passes through and scatters in different directions. The degree of scatter is captured by detectors...about the cell. Light that continues in the same direction as it was initially traveling is called forward...against a target is conjugated to a fluorochrome. In indirect staining a primary antibody binds to a target ...

... live on set, but peering at a 7-inch wireless monitor. Wireless video technology has gotten very impressive...   Figure 4: The remote directing setup, complete with wireless video receiver, camera control,... we decided to focus on a protocol that didn’t require any lab shooting, just animation. The plan was ...to shoot everything from her home, while I would direct “virtually” via a webcam. In the office, we use...this a lot easier and I didn’t need to be there directing. Everyone’s schedules were busy, so it was important...playing over the voice-over in videos), I sent some directions and (rules) to some volunteers, who filmed with... that I wanted to be able to talk to people and direct them. For interviews, being able to ask follow ...

...terminator sequence), since this would directly compete with the required single-stranded annealing and priming...modular overlap-directed assembly with linkers) which brings modularity to overlap-directed methods (Casini...they can all work at the same temperature, so the entire reaction takes an hour or less to complete at 50... resulting in scar-free assembly that does not require the presence of specific restriction sites (or ...sequentially.   How does Gibson assembly work? The required homology between neighboring fragments can be ...be because the exonuclease could chew through an entire fragment shorter than 200 nucleotides before the...techniques described above, this resulted in the direct and seamless cloning into a vector where no other...

... neurons projects axons directly to the prefrontal cortex, but also indirectly via synapses to neurons...dyes usually spread unidirectionally, there are some reports of bi-directionality. For predominately anterograde...specifically AAV6 in the retrograde direction and AAV2 in the anterograde direction (Salegio et al., 2013). Zingg...E.M., Mu, D., Hudry, E., Hyman, B.T., Maguire, C.A. and Ramirez S.H. (2017). Trafficking of adenoassociated...autobiographical events (see Jin & Maren, 2015). Directional flow of information between different parts of...for tracing compounds to outline neurons in the direction of information flow in order to understand which...retrograde tracing outlines neurons in the opposite direction, from the terminals of their axons to their cell...

...experiments can require steps such as protein expression and purification, which will require additional laboratory...being developed. Performing a CRISPR experiment requires delivery of, at minimum, two components: the Cas9...occur due to prolonged Cas9 expression     The requirement for Cas9 transcription and translation delays...which avoids many of these complications, is to directly deliver a ribonucleoprotein (RNP), consisting ...variety of cell types, gene editing using homology directed repair (HDR), and generating large genomic deletions...are not expressed. Because this method does not require the delivery of foreign DNA, and the Cas9-gRNA ...applications where limited expression of Cas9 is required and specificity is a concern, such as knockout...

... endeavor, NEBuilder requires upfront planning. This cloning methodology requires that the fragments to...multi-position site-directed mutagenesis by assembling multiple fragments containing the desired mutations. This...homology and any desired edits to the target region. Assembled plasmids can be directly transformed into...from New England Biolabs® has minimal upfront requirements and allows you to expediently join multiple ...DNA assembly, and allowing fragments to be taken directly from restriction enzyme digests to DNA assembly...plasmids via restriction enzyme digestion, and used directly in DNA assembly will still yield scarless assemblies...plasmids by gene here), designing primers with your desired mutation, performing PCR to generate the mutation...

...depolarization of neurons upon exposure to light through direct stimulation of ion channels. Naturally occurring...genetically-encoded nuclear Cry2 fusion can thus direct a CIB1-protein fusion to the nucleus upon light... tools and improve upon previous tools through directed mutagenesis - use Addgene's Optical Switch Plasmid...Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nat Methods...