...same overall process. First users coat a multiwell plate with a capture reagent. The capture reagent may ...substrates since they can be read on a standard plate reader as opposed to chemifluorescent, and chemiluminescent...antibody binds to antigen coated on the multiwell plate. In an indirect ELISA a primary antibody first binds...primary antibody. In a sandwich ELISA the multiwell plate is first coated with a capture antibody that binds...can be used. In a competitive ELISA a multiwell plate is coated with a competing antigen (green).  The...labeled antibody and then added to the multiwell plate. The more antigen in the sample, the less antibody...the sandwich ELISA, users first coat a multiwell plate with a capture antibody and then add their antigen-containing...

...the blocking-only templates will compete with the blocking/intended mutation templates resulting in cells...simultaneously introducing a homologous DNA repair template, such as a sin­gle-stranded oligodeoxynucleotide... very excited to set up a CRISPR-based editing platform in our lab. Our goal was to use the system to ... – Edit the cells a second time using a repair template that corrects the blocking mutation and again ...that the cell was only using a part of the repair template and mutations further away from the cut site were...intended mutation site are available, a repair template containing blocking and intended mutations can...can be equally mixed with another ssODN template that contains the same CRISPR/Cas-blocking mutation but...

...of cells. The antibiotic in your plate might not work. Check the plates! Streak on them an E. coli  strain...Design primers that overlap at least 24 bp with the template and if the sequence has high GC or AT content,...if you are having difficulties in amplifying a template, particularly from a large genome, you can recheck...have no colonies, check that the antibiotic in the plate correspond to the antibiotic resistance marker present...growing fine, it is probably time to prepare new plates with a fresh stock of antibiotic. Many small colonies...surrounding big colonies. You probably left your plate for too long in the incubator. The small colonies... different temperature (30°C) for the growth on plates and in liquid culture. If you don’t get any positive...

...proteins within a 96- or 384-well, flat-bottomed plate. The basic principle of an ELISA is that any antigen...works by first coating the wells of a flat bottomed plate with a capture antibody that is specific to the ...experimental sample are bound to the wells of a plate instead of an antibody. Coating the wells with a...characterize antibodies against the antigen bound to the plate. Figure 3: Overview of using Direct ELISA...protein antigen is coated onto the wells of the plate. However, instead of detecting your protein using...absorbance of each well, which is measured using a microplate reader, is directly proportional to the amount...your experiment. These are wells on the same ELISA plate that include known concentrations of your protein...

...to the template strand. Therefore, I suggest that picking a crRNA complementary to the template strand...tagging experiments by assembling csRNPs in a 96-well plate format and tested parallel tag knock-in for 96 different...single go (Figure 3). We were able to tag >30 genes/plate in the first shot with typical knock-in efficiencies...knock-in experiment were pre-assembled in a 96-well plate. Glioma stem cells were transfected in 96-well format...device; 5 days after the transfection, the entire plate was scanned for V5 tagging using an Operetta imaging... Clarke et al shows that crRNAs binding to the template strand (strand used by RNA polymerase II for transcription...e.g. convert the NGG PAM to NGC in your repair template) and use homology arms of 60 -70 bp . Purchase...

...confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive colonies...for example for subcloning genes from different templates. All PCRs are run as an 18 cycle, 25 μl single-tube...methylated DNA, so it will selectively destroy the template, but not newly synthesized sequences, limiting...Primers consist of two regions: the 3’ end binds template DNA for PCR amplification, while modifications...sequences are encoded at the 5’ end. First design the template binding region, with a melting temperature (Tm... bp, such as epitope tags, design primers with template binding regions to amplify away from the insertion... frozen: just defrost and add your primers and template. Amplifying the ‘unamplifiable’ Check your amplification...

...up your plate in a way that will tell you if you have pipetting errors on the edges of the plate. Compare...variation due to technical error.  Plate readouts can be affected by plate type or tubes, sealing method, ...possible to multiplex your reference genes to save on plate space. For many cell lines and tissue types, appropriate...quantification. A consistent 5% positive error across the plate is fine; a 5% positive error on one side and a 5%...reagents can prevent your reaction from reaching the plateau stage after exponential amplification, making it...multiplexing your reference gene set can save significant plate space and may be worth the initial effort of setting...to set the channel wavelength before you run the plate.    Analyzing There are a number of methods...

... the then RNAi Platform, which has since evolved into the Genetic Perturbation Platform and encompasses...Associate Director of the Genetic Perturbation Platform at the Broad Institute and has worked with many... up on a Saturday at like 7 am to go check your plates and you’re excited to do so, you know that that...The model that the Broad was building that had Platforms with a capital ‘P’ focused on technology ... has...enjoys working on technology development under the Platform model at the Broad Institute because they are ...CRISPR Learn more about the Broad Institute and the Platform Model. ...

...Addgene, we typically load 3 dilutions onto our ddPCR plate. As the titer is unknown, each dilution should fall...making the dilutions, they are transferred to a new plate containing the mastermix which includes primers ...a droplet reader extracts the droplets from the plate and measures the fluorescence amplitude of each ...positive (blue) and negative (gray) droplets. The no template control wells should have very few positive droplets...approximately 1 positive copy per microliter in the no template control wells. You’ll also notice a reduction ...sensitive assay, so cross contamination into a no template control (NTC) well is common. Here are some steps... droplets from the droplet generator to the PCR plate. Maintaining a high droplet count is important for...

... will guide the cell to use the donor DNA as a template for repair, which will result in the incorporation... knock-in! So how does the donor get used as a template for repair? Read on to find out! Homology-directed...basis of the HDR pathway relies on repair from a template molecule, which is usually an endogenously available...and uses the ends of the ssDNA to find a repair template. The highest HDR efficiency is observed when inserts...making the cut off essentially as part of the templated DNA. Check out our blog post on increasing HDR...To make sure your donor molecule is used as the template, you need to incorporate homologous sequence to...sister chromatid is likely to exist as a repair template. To maximize HDR events, ensure cells are actively...

...one AAV and a repair template in second a second AAV to deliver their repair template to mouse zygotes and...homologous recombination with a homologous repair template. It is this application - the creation of sequence-specific...allowing a researcher to fill it with the repair template. Bok and Porteus 2017 recently showed that by ... as RNPs and delivering two halves of a repair template spread across two AAVs, it is possible sequentially...this technique, Bok and Porteus deliver a repair template that would take up ~6.5 kB if delivered in a single...technique to efficiently deliver large repair templates for applications that require large gene fusions...

...with ssDNA repair templates. Their work demonstrates that different repair template strategies activate...using dsDNA templates achieving ~1-10% efficiency. Easi-CRISPR uses a long ssDNA template to increase ...working with CRISPR than just designing a repair template that contains your desired edit, and I want to...Editing in Human Cells Hands-on tips for repair template design from guest bloggers Dominik Paquet and ...

...individually generate gRNAs and homology arm repair templates containing the tag for each insertion. Also, it..., as well as the 5’ and 3’ homology arm repair template (Fig. 1, Step 1). There is no need for PCR or ...the second site accepts the homology arm repair template. SapTrap includes a prebuilt donor plasmid library...be used to generate functionally unique repair templates. A final ligation reaction correctly assembles...genetic locus, the toolkit also offers repair templates for tagging proteins in a tissue specific manner... and repressors, can be included in the repair template for inducible alteration of protein expression...

... science.                    Two of the platforms we use at Addgene: YouTube, for longer educational...octogenarians is going to be a hard sell!) Digital platforms often have demographic data of their users readily...geographic location. For example, not all social media platforms are available or used in all countries; not all...the limitations and potential of your preferred platform before you start can save a lot of trouble in ...requirements.  No visuals. Variety of hosting platforms can splinter audience. Primarily digital distribution...communicators. Well-developed content creation platforms.  Algorithms can make it difficult to reach people...

... to develop simple, portable, and inexpensive platforms to reliably detect nucleic acids at the atomolar...point, they hope that the protocol serves as a platform for advancing diagnostics.     DETECTR- DNA...harnessed this ability to create a DNA detection platform called DETECTR. How does DETECTR work? DETECTR...technology with the mission to “provide a CRISPR-based platform on which an infinite number of tests” for biosensing...Biosciences and UCSF recently adapted the DETECTR platform to detect SARS-CoV-2 using a lateral flow strip...Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360:439–...

...promoter or the coding sequence and the template or non-template strand, among other parameters. .  Stanley...introduced along with an oligonucleotide repair template. Through recombineering, the locus of interest...interest is modified to match the repair template, and the locus cannot be recognized by the spacer-derived ... contains the specific gRNA(s), and the repair template is supplied as a dsDNA fragment. Each round of...with XbaI to insert extra elements, like a repair template, using Gibson Assembly or restriction enzyme cloning... WA (2013) Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression...

...assembly cloning platform called GMAP (Gibson assembly-based modular assembly platform). GMAP is based ...orphan GPCRs demonstrating the utility of this platform for drug discovery. Researchers can use plasmids...Kit are available as glycerol stocks in a single plate with every plasmid or as individual plasmids shipped...Gateway cloning and the FNLCR Combinatorial Cloning Platform (CCP); the latter of which greatly enhances the.... PubMed PMID: 26165496 GMAP - A new assembly platform for generating both simple and complex DNA constructs...capabilities of Gibson assembly and make a more modular platform, the Jacks lab generated 5 common 30bp overlap...However, the laborious construction of repair templates and guide RNA constructs, as well as the difficult...

...antibiotic resistance marker. After transformation, plate the cells on medium containing the corresponding...contains a kanamycin resistance cassette, you would plate the cells on kanamycin-containing agar. Only cells... are auxotrophic for specific amino acids. When plated on media lacking the essential amino acid, the ... a toxic metabolite in gram-negative bacteria. Plating on sucrose medium will select for cells that contain...gene is used for negative selection on 5% sucrose plates. Exchange of the sacB gene for the gene of interest...

...DNA from each well of a 96-well plate. Then, samples from several plates are pooled together to create ...samples individually, plexWell™ reduces every 96 well plate to a single tube after the first tagging step. As... samples (e.g. a collection of 96 plasmids on a plate) will typically have a wide range of input quantities... from plexWell™ NGS data. Once Addgene produces plates of plasmid DNA, they are shipped to seqWell, where...

...devices for research. There are a number of new platforms (such as openUC2, which I am a co-founder of),...that make everyday microscopy much easier. On platforms such as Github, users can download the software...example, can also be printed quickly in Madagascar. Platforms that allow for on-going creativity with the construction...organizations and companies to provide problem-solving platforms.     Fig. 3: Different workflows involved...Prakash, "Squid: Simplifying Quantitative Imaging Platform Development and Deployment." bioRxiv. 2020. Diederich...