...applications common to each category, including an outline of the basic steps needed for each application, important...different isotypes for each primary antibody and secondary antibodies specific to each isotype. Overview of...confusing data. Check that your machine has appropriate lasers and filters for each fluorophore you select. Suggested... itself prior to antibody production; therefore, each B cell only produces one type of antibody. Monocultures...enable higher production of a single antibody, but each B cell can only produce one variable region. Isotypes...-antibody units, while others exist as monomers. Each antibody class therefore has a valency, or number...typically the number of antibodies multiplied by two, as each antibody has two binding sites. Isotype Description...

...using a sequencing machine that recognizes the different fluorescent labels. The machine detects which fluorescently...templates, are spatially separated from each other. These attached DNA templates are then amplified again...again producing ~1,000 copies of each template. Each template is then replicated using the modified bases...fluorescent color that is emitted each time a base is added. Again, each base (A,C,T, or G) is labelled ...an organism. Proteins are made up of amino acids. Each amino acid is encoded for by 3 nucleotides termed...20 natural amino acids and 64 codon combinations each amino acid is encoded for by multiple codons. Plasmids...fragments, which is far too many to separate from each other for the purposes of isolating one specific...

...technologies. Before CRISPR, genome engineering approaches like zinc finger nucleases (ZFNs) or transcription-activator-like...rapidly became the most popular genome engineering approach. Figure 1: Overview of the basic CRISPR mechanism...the ways the CRISPR components are leveraged to achieve specific goals, such as targeting multiple genomic...gRNAs into a single plasmid — though some have achieved targeting in the double digits. These multiplex...nickase system is utilized in many CRISPR systems to achieve specific genetic edits. The dCas9 molecule binds...termed left and right homology arms). The length of each homology arm depends on the size of the change being...system, the Cas effectors lack nuclease activity. Each system also includes Tn7-like transposase components...

...) until peak expression of the opsin is reached. This approach is especially powerful when combined with...including a brief overview of the wild-type opsin of each and some examples of relevant variants. This list...closing of the channel 470(activ.), 590(inact.) ReaChR Red-activatable variant of CrChR2 590 Channelrhodopsins... variant 467 Aurora Red-shifted anion-selective ReaChR variant 517 Halorhodopsins Jaws Red-shifted, light-driven...Generally, the most robust and stable expression is achieved in a transgenic mouse line, such as for example...that bind to the LOV domain only in the dark. attaching only one member of the Zdk/LOV2 pair to the target...KR, Deisseroth K. 2010 Molecular and cellular approaches for diversifying and extending optogenetics. ...

...specificity. Dual-nickase approach increases specificity but is less efficient. Each putative knockout allele...reagents may be sufficient to express the CRISPR machinery. For more difficult cells (e.g. primary cells)...result in reduced cleavage. The number of alleles for each gene may vary depending on the specific cell line...essential protein domains. The benefit of this approach is that even non-frameshift alleles may alter ...Furthermore, in cells containing two mutated alleles, each mutated allele may be different owing to the error-prone... examine off-target effects. More information on each of these techniques can be found in our blog post...

...move around pieces of DNA. Oftentimes several approaches will work for any specific cloning project; however...likely that for any given project there is an ideal approach. This may be due to speed, cost, availability ... of time required for cloning. To utilize this approach, the fragment of DNA that you would like to clone...amplified with specific Gateway attB1 and attB2 sites attached to the 5’ and 3’ ends of DNA sequence. This fragment...fragments generating long overhangs that anneal to each other due to their homology. DNA polymerase then... enzyme cloning. Scientists have utilized this approach to create compatible custom overhangs that can...

... packaging genes and accessory genes specific to each virus type, while retroviruses only contain packaging... thousands of target genes and require gRNAs for each target. Lentiviral vectors are a common way of delivering...doi.org/10.1126/science.272.5259.263 PMID: 8602510 Schambach, A., Zychlinski, D., Ehrnstroem, B., & Baum, C... (1997). Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nature Biotechnology...comprised of a U3-R-U5 structure and are found on each side of the provirus. The U3 (unique 3’) contains...

...replication as it activates the necessary cellular machinery to produce large amounts of rAAV vectors. The ...molecular weight structures that are maintained extrachromosomally. Random integration of rAAV genomes in non-homologous... vector depends on the cell’s DNA replication machinery to synthesize the complementary strand, transgene... Having the shape of a polyhedron with 20 faces, each being an equilateral triangle. Immunogenicity The...replication process where the virus uses the cell machinery to produce new viruses, eventually lysing the ...

...cDNA libraries, each plasmid contains a unique cDNA. In shRNA or gRNA libraries, each plasmid contains...sequence, but there are multiple sequences targeting each gene in the overall library. Barcoding libraries...negative results - for a given plasmid, think of each cell carrying it as being a biological replicate...

...engineered α7 nicotinic acetylcholine receptor (nAChR) domains that respond to specific small molecules...Adeno-associated virus (AAV) is a widely-used tool for achieving in vivo expression of chemogenetic receptors, ... our AAV guide . FLEx Vectors are also used to achieve cell-specific expression of AAV-encoded chemogenetic...Azua IR, Li JH, et al. (2009). A chemical-genetic approach to study G protein regulation of β cell function...

...lentiviruses also contain accessory genes specific to each virus type. From an experimental standpoint, the... 24464997 Maetzig, T., Galla, M., Baum, C., & Schambach, A. (2011). Gammaretroviral vectors: Biology, ...comprised of a U3-R-U5 structure and are found on each side of the provirus. The U3 (unique 3’) contains...

...into the host genome. Instead, they remain as extrachromosomal elements within the nucleus, normally as double-stranded...have been developed to reduce immune response and achieve longer transgene expression. Adenoviral Types Serotypes... Having the shape of a polyhedron with 20 faces, each being an equilateral triangle. Immunogenicity The...

...guide to viral vectors contains an introduction to each vector and its applications, tips for using vectors...

...RNA polymerase in binding to the promoter region. Each sigma factor recognizes different core promoter ...