... vector, contains a promoterless splice acceptor-IRES-neoR gene, which enhances the efficiency of homologous...applications that require simultaneous modification of all alleles of a gene, or which require modifying multiple...experiment. For this reason, the AAV-based approach requires G418 selection of infected clones, making it unsuitable...suited for tissue culture based approaches that require modification of only a single allele of a gene,...deliver large repair templates for applications that require large gene fusions. The current state-of-the-art...PMCID: PMC4752446. 5. Asuri, Prashanth, et al. "Directed evolution of adeno-associated virus for enhanced...

...Simply cotransfect a mixture of 1248 ng of CMV-gvpA-IRES-BFP plasmid and 954 ng CMV-gvpNJKFGWV plasmid into...booster cassette), their expression heterogeneity required single cell screening to isolate a clonal cell...producing ultrasound contrast, and gene silencing required addition of chromatic-opening agents to boost ...contrast. It turned out that Anabaena mARGs did not require a booster cassette, either, allowing us to reduce...marked with Emerald GFP. These plasmids can be used directly in transient transfection in rtTA+ cell lines ...

... fluorescent protein downstream, separated by an IRES element. You did a lot of work! But let’s take a...-thi-1 or thi1 Mutation in thiamine metabolism Requires exogenous thiamine for growth ara disruption...leuB β-isopropyl malate dehydrogenase inactivated Requires exogenous leucine source for growth proAB mutation...mutation in proline biosynthesis pathway Requires exogenous proline source for growth rpsL Mutation ...This strain is endA+, so column-based DNA preps require an additional wash step. F- glnV44 recA13 mcrB ...

.../hum.2019.277 Lavin TK, Jin L, Lea NE, Wickersham IR (2020) Monosynaptic Tracing Success Depends Critically...Many neuroscience experiments that require gene expression in a specific cell type rely on transgenic...lox2272, that are in the opposite orientation (direction of arrows) the inverted transgene is reverted ...promoters, such as CAG are very strong and may require less viral particles or relatively short expression...such as hSynapsin, are relatively weak and may require longer waiting periods and/or higher injection ...recombination rate (0.1% and 0.13% respectively) still required a 200-300 fold dilution to minimize off-target...Conclusion Using AAV vectors in your experiments requires a full understanding of vector performance in ...

...while wavelengths over 700 nm make up the infrared (IR) spectrum. When photons are absorbed by matter, they...

...four types of ELISA are direct, indirect, sandwich and competitive. In a direct ELISA a reporter enzyme...competing antigen. In direct, indirect and sandwich ELISA the reporter signal is directly proportional to the... the bench.   Indirect ELISAs The indirect ELISA process is similar to that of a direct ELISA, but the...figure was created with BioRender.com  Direct ELISAs A direct ELISA is the quickest and simplest of all...source appropriate antibodies for an indirect ELISA than for a direct. In addition, a lab can use the same...available to bind to the reporter. Unlike for direct, indirect, and sandwich ELISAs, in a competitive ELISA...are similar to those of the direct method. Namely, since the assay requires a single conjugated primary...

...all use either the direct approach or indirect approach, depending on the requirements of the specific assay... also be. This is the direct approach to immunoimaging assays. But if the direct approach isn’t giving... associated, indirectly, with each protein of interest.    Figure 1: The direct approach (left...always use the indirect approach, while flow cytometry’s standard approach is the direct method. ELISAs...secondary antibodies require their own incubation and wash steps, which means that indirect methods are often...sometimes assumed that the direct method is more quantitative than the indirect method. But in reality, ...quantification can be done using either the direct or the indirect method.  New Methods Time is a precious...

...sample, whereas antigen is bound directly to the wells in a direct and indirect ELISA.   Sandwich ELISA Most...et al., 2019.   Indirect ELISA The indirect ELISA is very similar to a direct ELISA, aside from the...detected. These are sandwich, competitive, direct, and indirect. Sandwich and competitive ELISA use antibodies...the sandwich ELISA as described here, the direct and indirect ELISA (more details below) can also be adapted...from Boguszewska et al., 2019.       Direct ELISA In a direct ELISA, purified proteins or the experimental...for visualization. The indirect ELISA is used for similar experiments as the direct ELISA, the only difference...Antibodies are used in many different experiments that require scientists to detect proteins in their samples....

...deliver substances directly to the lungs. It is an advanced technique, as it requires intubation of the...< 0.05 ml Skill required: beginner Oral gavage Oral gavage delivers drugs that require ingestion, such...after initial mouse training. Other techniques require more skill and additional training, going from ...seem like the easier of the two, but it actually requires more technical skill to place the needle in the...is slower, and more institutional oversight is required. Restraint: mechanical restraint or anesthesia...-orbitally Gauge: 27–30 Volume: < 0.2 ml Skill required: beginner (retro-orbital) to intermediate (tail...Like IV injections, intraperitoneal injections require a little bit of practice to ensure the proper injection...

...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...

...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...

...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...

...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...

...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...

...crRNA/gRNA length gRNA: ~100 nt crNA: ~42 nt Requires tracrRNA? Yes No dsDNA cleavage Blunt end 5'... work with a transfectable cell line. 2) They require larger expression vectors which are often more ...Cas9 multiplexing vectors are larger because they require regulatory sequences to allow for expression of...vector expresses Cas9 and up to 7 gRNAs Each gRNA requires its own promoter Sakuma et al   Multiplexed...expresses Cas9, eGFP, and up to 4 gRNAs Each gRNA requires its own promoter   Kabadi et al Csy4-Cleavable...gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9 Nissim et alTsai et al...gRNAs expressed from a polycistronic transcript Requires co-transfection of Cas9   Xie et al  Multiplexing...

...components are required? CasPEDIA provides information on crRNA and tracrRNA requirements, along with any... any other required non-CRISPR-associated host factors required for CRISPR processing. For each enzyme...classification system, CasID, to help facilitate direct comparison of different enzymes. CasPEDIA is meant... properties (CasID): Cis/trans activity, PAM requirement, gRNA, and multiplex ability Experimental guidelines...a candidate Cas for your experiment. Target requirements As we all know, a Cas guided by a gRNA is useless...) in some cases. The resource displays these requirements in regard to their sequence (NGG, etc.) as well... gRNA (5’ or 3’). In some cases, there is no requirement, which is also documented. Experimental design...

...biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous end-joining (NHEJ) pathway...sequence change the cell must undergo homology-directed repair (HDR), a distinct cellular DNA repair pathway... is relatively rare and DSBs are predominantly repaired by NHEJ. While other groups have focused on strategies...overall HDR rates, we have shown recently that desired genome-editing events by HDR can be generated more...introduced. Although we were able to introduce the desired mutations in genes associated with dementia, such...CRISPR from re-cutting the target sequence once the desired edit has been introduced. We found that blocking... or guide RNA blocking mutations? This depends entirely on the locus you are editing, and if the blocking...