...,5'-tetramethyl-[1,1'-biphenyl]-4,4'-diamine) substrate kit, Pierce 34021 Tween-20, VWR 0777-1L Foil Before...Solution and 5 mL Peroxide Solution from the TMB substrate kit. Cap the tube and invert several times to ...secondary antibody for 2 hours, reacted with TMB substrate and the reaction stopped after 15 minutes. Absorbance...

...function. We specifically use this protocol with a subclone of HEK293T cells optimized for viral production...Workflow Timeline Day 0: Seed HEK293T cells (or a subclone of HEK293T optimized for viral production) Day...

... lower the pipette tip into the liquid. Do not submerge the pipette itself into the liquid and avoid touching...contaminating the contents. Once the end of the tip is submerged, slowly release the plunger, allowing it to go...

... a water bath at 60 ℃ with sufficient water to submerge ~75% of the bottle containing your molten gel ...removing anything from the autoclave. Partially submerge your molten gel-mix in the 60 ℃ water bath. Pro-Tip...

... thought on using selective medium throughout subsequent experiments. When selective medium is used, it...because the buildup of waste products could be suboptimal for cell growth. This conditioned medium will...

...genes, and is unique in that it is composed of two sub-pools. The first is a base “90k library” that contains...using CRISPR-X and subsequent FACS sorting. They also mutagenized proteasome subunit PSMB5 to find mechanisms...Every quarter we highlight a subset of the new plasmids in the repository through our hot plasmids articles...mRNA binding, and these sequence changes are subsequently identified through transcriptome sequencing.... Guler Lab engineered an actuator in which two subunits of the paramagnetic ferritin protein were tethered...channel). The Guler lab optimized the actuator’s subcellular localization using trafficking signals and named...

...can be converted into lentiviral particles for subsequent infection of a target cell line. Once introduced...toxic to some cell lines. In these cell lines, substitute protamine sulfate for polybrene. c. Add lentiviral...

...secretory proteins or proteins targeted to membrane sub-domains. It has also been used to observe intra- ...Renilla luciferase, catalyzes the oxidation of its substrate, luciferin, which releases energy as a photon.... shown to be useful tools for light-mediated subcellular localization in mammalian cell culture and reversible...were used in a variety of experiments including subcellular visualization of 6 different organelles, a cell...photoactivatable fluorescent proteins (FPs). A subpopulation of mitochondria is selectively labeled by irradiating...assaying the dynamics of different mitochondrial subpopulations within the same cell; by photoswitching PSmOrange...PSmOrange and photoactivating PAGFP in separate subpopulations of mitochondria, one can monitor how the two...

...stable for years, as long as it is kept at -80°C. Subsequent freeze and thaw cycles reduce shelf life. To ...

...with an empty tube. Alternatively, you can just subtract the weight of the empty tube from the weight of...

...weighing and prevent cross-contamination with other substances. They also help you transfer the material to ...

...buffer) to ensure optimal salt concentrations in subsequent reactions. Step 3: Create Vector Overhangs Treat...

...vary between cell lines. It is not recommended to subject lentiviral supernatants to multiple freeze-thaw...

... DNA 5’ end. Complementary DNA fragments can subsequently anneal to each other. Phusion High-Fidelity ...

...not recommended that lentiviral supernatants be subjected to multiple freeze-thaw cycles. This protocol ...

...deposit process by clicking "Submit Online Deposit" (for online submission) or "Download Deposit Spreadsheet...emailed submission) in the Deposit menu: Figure 1: Begin the deposit process by clicking "Submit Online...    Figure 3: If submitting plasmid(s) online (left), choose to submit published or unpublished...page and click the blue "Submit Deposit" button. Figure 13: Click the "Submit Deposit" button to complete...begin your plasmid deposit. Figure 2: Choose to submit plasmid(s) online or by using a spreadsheet. The...Fields marked with an asterisk are required. If submitting plasmid(s) using a spreadsheet (right), click...download the .xls file. For the online form you can submit "Published plasmids" or "Unpublished or pre-publication...

...recombineering substrates to avoid errors introduced during PCR. ssDNA Substrate ssDNA substrates can be generated...   Substrate DNA Design and Generation Whether you use a linear dsDNA or ssDNA substrate depends ...ssDNA substrate is best for point mutations or changes of only a few base pairs.  dsDNA Substrate dsDNA...Pro Tip: ssDNA substrates have a much higher rate of recombination than dsDNA substrates, so you should...DNA, you first electroporate a linear donor DNA substrate (either dsDNA or ssDNA – see below) into E. coli... catalyze the homologous recombination of the substrate with the target DNA sequence. This means cloning...genetic changes occur in a test tube. The donor DNA substrate only requires ~50 nucleotides of homology to the...

...blog post shows. To avoid these issues, Subach et al. and Tsuboi et al. developed monomeric FP timers with...monomeric FP timers (mK-GO from Tsuboi et al. and the FT series from Subach et al.) were derived from previously...membrane protein, and following its subcellular localization overtime (Subach et al.) Monitor protein expression...tetramer with a propensity to aggregate in cells (Tsuboi et al.), limiting its use. Nonetheless, its creators...fluorescent proteins mKO and mCherry, respectively. While Tsuboi et al developed mK-GO somewhat serendipitously ... while attempting to enhance mKO in other ways, Subach et al made a concerted effort to develop their ...see Table 1 for emission and absorption spectra). Subach et al’s directed mutagenesis also produced the ...

... out Cas3. Type I is further subdivided into subtypes A–G. These subtypes have similar compositions and...immunity. Within type III, there are six subtypes, A–F. All subtypes use Cas10 as the primary effector complex...into a fairly simple hierarchy: class to type to subtype to variant. Despite the seemingly complicated steps...types are based on main function and composition; subtypes and variants are typically based on species of...There are currently two classes, six types, 33 subtypes, and 17 variants. Figure 1: Overview of...hierarchy. The hierarchy flows from Class to type to subtype to variant (shown in italics). Created with BioRender.com...Figure 2: Schematic of a Cascade complex (blue subunits) with a guide RNA (green and orange) bound to ...

...not recommended that lentiviral supernatants be subjected to multiple freeze-thaw cycles. Procedure Before...