... Lin28A to somatic cells from separate plasmids Highly Efficient Reprogramming to Pluripotency and Directed...

...reagent you will be weighing out, a weighing boat or weighing paper to contain the reagent while on the...that you are weighing by looking for a weight range on the scale. Make sure that the weight of the material...out the material you are weighing onto the weighing boat or paper. If you weighed out too much material,... up. Discard the weighing boat or weighing paper in the trash if the material weighed is not biohazardous... Protocols Weighing Reagents Weighing Reagents Protocol Intro ... to handling the materials. Equipment Weighing boats or weighing paper Balance Scoopula or spatula Container...button. Before weighing out your reagents, determine the amount that you need to weigh. Gather the reagent...

...The route the light follows from the source to your eyes is called the light path . Light travels from ... Protocols Using a Light Microscope Using a Light Microscope Intro ... to encounter: the compound light microscope. As the name suggests, light microscopes take advantage of...compound light microscope with the main components labeled: Figure 1: Diagram of a compound light microscope...from the light source through a condenser that helps concentrate the light onto the sample, and then through...objective! Once on the higher objective, you will likely need to re-adjust lighting and focus. Increase ...basics of how to use a light microscope. Protocols...

...coronavirus using CRISPR-based SHERLOCK (Specific ​H​igh ​s​ensitivity ​E​nzymatic ​R​eporter un​LOCK​ing)... under either no excitation light, UV light or LED blue light.  Highlights: This is a one-pot, one-temperature...heat block, LED blue light illuminator or UV light illuminator. Approach: Highly similar to the SHERLOCK...captured by an antibody further along on the strip. Highlights: Of the 83 known COVID-positive samples tested... extraction but that may work only if there is a high enough (105 copies per microliter) viral load in...lateral-flow readout slip that allows rapid detection.  Highlights: Cas13a is not catalytically active in the presence... duplex and will not give a positive result for highly similar viruses. Unlike Cas12a, Cas13a does not...

...Microbial Opsins Opsins are light-gated ion channels or pumps that absorb light at specific wavelengths. ...respond to different wavelengths of light, like blue or yellow light. These various opsins also initiate...also a blue light activated cation channel. ChR2 is preferred over ChR1 because ChR2 has higher conductance...Channelrhodopsin from Volvox carteri 570 Chronos High-speed, light-sensitive channel from Stigeoclonium helveticum... 566 ArchT Light-activated outward proton pump from Halorubrum strain TP009. Improved light sensitivity...binding domain, allowing light to activate transcription. Phytochromes and light oxygen voltage (LOV) domains...) BLUF domains bPAC Light-activated adenylyl cyclase from Beggiatoa sp. for light-induced cAMP modulation...

...the Fab fragments contain highly variable regions, shown in light blue and light orange, that bind to a ...are light-sensitive molecules which emit a specific wavelength of light after excitation by light of a...pair the brightness of the fluorophore with the abundance of the antibody target. If a bright fluorophore...molecular tools used in the lab to specifically and tightly bind to other proteins. Antibodies, produced as...-efficient way to produce antibodies, with the highest variability in antibodies produced. Cells - Antibodies...protein chains: two large heavy chains and two small light chains. These four domains come together to form... to be produced in a single individual, giving a high probability that an antibody will be able to recognize...

...joining (NHEJ) pathway and the less efficient but high-fidelity homology directed repair (HDR) pathway....also enhanced specificity by engineering Cas9 into high fidelity Cas9s (hfCas9) . There is currently no ..., which are about the same size as SaCas9, with higher fidelity than SpCas9. FrCas9 (from Faecalibaculum... downstream of the target site (termed left and right homology arms). The length of each homology arm ...available with multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements...strands Prime editing and base editing both offer high efficiency and low indel formation compared to HDR...large-scale, forward genetic screening. CRISPR can make highly specific, permanent genetic modifications to eliminate...

...opto-chemogenetic fusion proteins of a light-emitting luciferase and a light-sensing optogenetic element. When...neurotransmission. This, and the fact that GPCR ligands can have high potency, which is potentially important for dosing...off-target effects in vivo and the receptors exhibited high levels of constitutive activity, making them less...luciferin) is added, the luciferase enzyme generates light that activates the opsin. The opsin may excite or...illumination), making LMOs a versatile option. Coupling brighter bioluminescent modules, such as luciferase-fluorescent...example, a study in rats using an hM4Di receptor might consider an alternative DREADD ligand such as DCZ... chemogenetics by using physical and biological light sources for opsin activation. Proc Natl Acad Sci...

...These episomes can concatemerize, producing high molecular weight structures that are maintained extrachromosomally... a hybrid capsid derived from eight serotypes. AAV-DJ displays a higher transduction efficiency in vitro...separation of Rep and Cap shown in the figure on the right facilitates the viral pseudotyping discussed below... of transgenes, as well as the expression of neighboring genes. The short coding sequences of channelrhodopsins...any wild type serotype; in vivo , it displays very high infectivity across a broad range of cell types. ...

...insert expression (i.e., a strong promoter directs high expression, whereas weaker promoters can direct ...determines the type of plasmid and dictates how it might be used in the lab. Below are some common plasmid...particles, that can infect your target cells at a high efficiency. Addgene's expanding viral service offers...Next Generation Sequencing (NGS) comes in. NGS is a high-throughput, multi parallel sequencing platform that... Plasmids 101: 5 factors to help you choose the right cloning method Plasmids 101: Restriction Cloning...

...will have the highest specificity. To increase specificity, you can also consider using a high-fidelity Cas...gRNA targeting 5′ exon or essential protein domains High-fidelity Cas enzymes increase specificity. Dual-...a mammalian cell line that can be transfected at high efficiency Lentiviral transduction Cas9 and gRNA...

... capacity of >8 Kb. Constructs contain left and right arms to facilitate homologous recombination of the...and the adenoviral plasmid have matching left and right homology arms which facilitate homologous recombination...the shuttle vector, but this method results in a higher background of non-recombinant adenoviral plasmids...

...choosing the right cloning method for your research project . The following guide will highlight several of...

...from Addgene. 2nd Generation The graphic to the right shows how the lentiviral genome is edited down and...lentivirus is outlined in the simple schematic to the right. 3-4 plasmids are transfected into A293T cells: ...however, they are different viruses and thus use slightly different isoforms of these packaging components...

...immune system, antibodies are proteins capable of highly specific binding to other proteins. Antibody-based...technologies developed for optogenetics research utilize light to detect, measure, and control molecular signals...

...however, they are different viruses and thus use slightly different isoforms of these packaging components...research to reduce promoter interference and/or drive high expression of a transgene from an internal promoter...

...additional hypothesis-directed experiments. As you might have guessed from their name, pooled libraries are...

...polymerase) to bind to a promoter. Transcription is tightly controlled in both bacteria and eukaryotes. Promoters...

...limit of a light microscope. Due to the wave nature of light, an infinitely small point of light will blur... to provide high 3D spatial resolution while also avoiding the toxic effects of high light doses to the...fluorescence microscopy provides a high contrast image in which a bright signal overlays a perfectly black...placed to only allow light from the focal plane to reach the detector and all light from above or below...for example, red light instead of blue to excite a molecule of GFP. Because red light has approximately...approximately half the energy of blue light, two photons of red light are needed to excite GFP compared to only ...objective can a a high enough density of red-photons be establish for this to happen.Using red light has a second...

...UV-Violet Dark/Green N/A (high) High 503/ 518 Padron Monomer Blue Dark/ Green N/A (high) Low 503/ 518 rsCherry...specific wavelength of light. PA-FPs can be activated from low fluorescent states to high fluorescence states...fluorescence, to a brighter fluorescent state by exposure to a specific wavelength of light. The switch or...fluorescence when illuminated with 488nm light. However, when UV-violet light is presented first, it causes irreversible...illuminated with violet light, both variants are photoconverted to forms that emit brighter red fluorescence....when exposed to blue light, which is less phototoxic to live tissue than the UV light used by other PA-FPs...State Activating Light Pre/ Post Color Contrast (Fold Change in Fluorescence) Brightness Excitation/ Emission...