...use the same restriction enzymes for cloning so multiple constructs can be made and tested at once. Return...

...Applications PI Additional Resources To learn more, please visit the BRAIN Initiative Resource List . NeuroMab...

...subclass from the conventional mAb to facilitate multiplex labeling with subclass-specific secondary antibodies...recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain. eLife . 8:e43322. PMID...

...antibody in assays with multiple targets, and is described in more detail in the Multiplex Assays section below...fragments (shown in Figure 2) to completely unrelated molecules. Examples include: scFv - Single-chain fragment...differentiation between multiple antibodies. They are often used in assays that have multiple proteins to detect...application Collect and lyse samples to make proteins available. Tissue samples may need additional processing...proteins from a complex mix. Overview of application Process the tissue or cell culture samples to extract ...capture protein:DNA complexes. After capture, the DNA is released from the complex and can be sequenced...culture samples to extract proteins. Homogenize (tissues only) and lyse. Cross-link the samples with formaldehyde...

... C RISPR- as sociated c omplex for a ntiviral de fense; a complex of multiple Cas enzymes including Cas8...edits carried out by Cas9. Multiplexing applications include editing multiple genes at once; using dual...detail later. Read more about Cas9 multiplexing . Browse Plasmids: Multiplex gRNA Vectors Engineering the Cas9...also has more condensed multiplexing capabilities , as it can process multiple gRNAs under a single promoter...can be added to SHERLOCKv2 to detect multiple targets. For example, Cas12 can be added to detect the presence...Cascade ( C RISPR- as sociated c omplex for a ntiviral de fense) complex to initiate DNA degradation. Cascade...have extended CRISPR applications to increasingly complex functions, including the ability to selectively...

...of each and some examples of relevant variants. This list is not exhaustive - please browse our curated...amplitude Examples: ChR2(H134R), C1V1(t/t), ChIEF Increased channel (on/off) kinetics: Examples: ChETA, ...Human codon optimization Example: Halo Increased photocurrent amplitude Examples: eNpHR, eNpHR2.0, eNpHR3.0...Increased light sensitivity Example: ArchT Increased photocurrent amplitude Examples: eArch3.0, eArchT3.0 Browse...make it possible to combine multiple opsins in the same experiment. For example, you could use different ... cells in order to understand their functions. Example schematic of an optogenetics procedure. A channelrhodopsin... variants to enhance the functionality of ChR. Examples of ChRs from other species include: CsChR (from...

...chemogenetic receptors were based on G-protein coupled receptors, or GPCRs. The largest class of cell ...chemogenetic tools. Need a refresher on G-protein coupled receptors? Read the GPCRs: How Do They Work and...like RASSLs, are based on engineered G-protein coupled receptors, but unlike RASSLs, show insensitivity...classified by the signaling protein that the receptors couple to: Gq-DREADDs signal through the Gαq/11 G-protein...is not widely used. There is also a DREADD that couples to β-arrestin to activate noncanonical GPCR signaling...For a discussion of alternative DREADD ligands, please see the text. Browse Addgene's collection of DREADD...allowed them to respond to their cognate PSEM. For example, PSAM Q79G,Q139G is activated by PSEM 22S , while...

...combinations each amino acid is encoded for by multiple codons. Plasmids and Recombinant DNA Technology... as cloned DNA, and this process of generating multiple identical copies of a recombinant DNA molecule... vary broadly in their functionality. In their simplest form, plasmids require a bacterial origin of replication...the bacterial host. Each bacterium can contain multiple copies of an individual plasmid, and ideally would...bacteria by bacterial transcriptional machinery. Multiple Cloning Site (MCS) Short segment of DNA which ... DNA fragments. Cloning vectors tend to be very simple, often containing only a bacterial resistance gene...elements. These plasmids contain a reporter gene (for example, luciferase or GFP) that offers a read-out of the...

...Gibson cloning is that it allows for the simple assembly of multiple fragments of DNA in the chosen orientation...ends in sequences that form complex secondary structues. You can view an example of a LIC protocol on our...overhang (blunt ends). Two pieces of DNA that have complementary overhangs, or which are both blunt-ended, can... same sites in the same orientation within its multiple cloning site . Due to their short length, it is...selection cassettes). Although gateway cloning is a simple and efficient cloning method, gateway vectors and...restriction cloning, once your project has been completed via gateway cloning. Back to Top TOPO® Cloning...method that relies on the hybridization of the complementary base pairs adenine (A) and thymine (T). TOPO...

...recommended to verify that the library is complete - an incomplete library may lead to false positives or...genes that contribute to a phenotype. At their simplest level, pooled libraries are single preparations... unique gene targeting sequence, but there are multiple sequences targeting each gene in the overall library...lineage tracing or parsing the effects of expressing multiple genes at once. A well-designed screen can help...your screen. If you need to amplify the library, please refer to the depositor’s protocol for the best ...number of plasmids present in the library - for example, the Moffat lab uses 200x the number of cells as...method relevant to the phenotype being studied. Examples of published selection mechanisms for lentiviral...

...as adenovirus or herpes simplex virus to replicate in the host cell and complete the lytic cycle. AAVs ...transduction efficiency and alter tropism. For example, AAV2/5, targets neurons that are not efficiently...Hybrid capsids are engineered capsids derived from multiple different serotypes, which can further alter transduction...transduction efficiency and viral tropism. One common example is AAV-DJ, which contains a hybrid capsid derived...blog post on AAVrg for more information. Self-complementary AAV (scAAV) One downside of the use of AAVs ...DNA replication machinery to synthesize the complementary strand, transgene expression may be delayed....capacity of rAAV vectors to ~2.4 kb. Figure 4: Examples of AAV serotypes, pseudotypes, hybrid capsids,...

... transcription is simpler than eukaryotic transcription bacteria still have complex systems of gene regulation...promoters, transcription and the transcription complex. Plasmid...other hand, the process in eukaryotes is much more complex. Eukaryotes require a minimum of seven transcription...Transcription Eukaryotic promoters are much more complex and diverse than prokaryotic promoters. Eukaryotic...away from the TSS. Eukaryotic promoters are so complex in structure that the DNA tends to fold back on...binding of the RNA polymerase. A transcription complex is constructed from the RNA polymerase and several...transcription. The Lac operon in bacteria is an example of a negatively controlled operon. A positive repressible...

... we will use the example of CRISPR/Cas9 in mammalian cells, many of these principles apply to using CRISPR... process of interest. Do you want to: Generate complete and permanent loss of gene expression or function...major concern, Cas9-gRNA ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression...transgenic embryos Cas9-gRNA ribonucleoprotein (RNP) complexes Purified Cas9 protein and in vitro transcribed...transcribed gRNA are combined to form a Cas9-gRNA complex, which is delivered to cells using cationic lipids Transient... will depend on your specific application. For example: To activate or repress a target gene using dCas9...nucleotides within the selected target sequence. For example, gRNA targeting sequences containing a G nucleotide...

...hence the use of “retro”. Lentiviruses have more complex genomes, containing packaging genes and accessory...along with virus-specific accessory genes (for example, vif , vpr , vpu , and nef for HIV-1). The LTRs...comparison of second- and third-generation systems, please refer to Table 1. Figure 4: Third-generation lentiviral...integration sites have led to the development of multiple genome-wide analysis pipelines to assess viral...Timms et al. (2018), and Wells et al. (2020) for examples). Pseudotyping Tropism dictates which types of...in the envelope and packaging plasmids. Unless multiple recombination events occur between the packaging.... (2021). CReVIS-Seq: A highly accurate and multiplexable method for genome-wide mapping of lentiviral...

...genomes range between 26–45 kb in length and contain multiple heavily-spliced transcripts flanked by two inverted...harvested about 7–10 days later. Vogelstein designed multiple shuttle plasmids for different purposes. The pAdTrack...that the infection rates are equivalent across multiple viruses. The adenoviral backbone plasmid pAdEasy...transduction efficiency, tropism and immunogenicity. For example, Ad5/35 and Ad5/26 combine the high transduction...packaging capacity. Large protein-coding genes and complex gene expression cassettes are often too big to ... also been developed as vaccine candidates for multiple infectious diseases and prostate cancer. Cancer...

... — hence the use of “retro”. Retroviruses have simpler genomes, containing only the necessary packaging...transfection step of the viral packaging cell line. For example, Phoenix™ is a popular second-generation gamma-...in the envelope and packaging plasmids. Unless multiple recombination events occur between the packaging...Hughes, S. H., & Varmus, H. E. (Eds.). (1997). Principles of Retroviral Vector Design. In Retroviruses ...

...for your convenience. For reference information, please consult Addgene's Molecular Biology Reference Page...vector, forward primer SFFV-F ATTGATTGACTGCCCACCTC Spleen focus forming virus 5' LTR, forward primer SP6 ...

...between the glass coverslip and a sample in aqueous buffer it is completely reflected. This reflection occurs...water-like buffer the sample is immersed in. Although the excitation light is completely reflected, energy...microscope to use for a new sample I always start by asking two questions: 1) Is the sample dynamic or static? ...? 2) Is the sample thin (< 15 μm) or thick? These questions will place each sample into one of four categories...capture in these types of samples is their fast movements. Depending on the sample, a temporal resolution... fluorophores higher in the sample. TIRF is also very gentle on the sample as the majority of the laser...sheets of excitation light through multiple focal planes of a sample may also be useful for providing fast...

...more complex experiments. A multiplexed experiment, for example, requires staining with multiple primary...control is generally a sample or group that will have a desired response. For example, if you are studying...to experimental samples (?). B, The inclusion of biological replicates such as multiple mice in each group...control is a sample or group that is not subjected to the experimental condition. In the example above, a ...inhibitors on mice, for example, it would be better to treat and test multiple mice with the drug rather... wild-type and knockout samples. In this test, wild-type and knockout samples are treated with the identical...present in the wild-type samples and absent in the knockout.  If knockout samples are unavailable, try substituting...

...numbers of plasmids. plexWell™ technology overview For any NGS experiment, the DNA sample being sequenced ...costly library prep, as each sample requires an individual library. The plexWell™ workflow is a sequential...carrying sample-specific barcodes. Rather than continuing to process 100s to 1000s of samples individually... of plexWell™’s sequential tagging procedure, the quantities of library molecules for each sample remain... problem in multiplex NGS library preparation. The challenge here is that, as more samples are sequenced...case that individual libraries prepared from multiple samples will then also need to be quantified and re-normalized...in order to be combined with other samples in a single multiplexed pool. The cost of these normalization...