... RNA amplification, new toehold switch designs, CRISPR/Cas9-mediated genotyping, as well as our electronic... synthetic gene networks for the detection of antibiotic resistance and strain-specific diagnostics for...electronic reader that provides portable quantification of diagnostic results.  Our approach now detects samples...

... genes for proteins in these compartments using CRISPR systems should strongly consider using moxFPs over...by guest bloggers Erik L. Snapp and Lindsey M. Costantini. "You underestimate the power of the Dark Side...non-fluorescent, and misfolded FP-fusions on quantitative imaging experiments and cell physiology in general...population is not readily apparent and can confound quantitative imaging experiments or even negatively impact...misfolded dark FP fusions including erroneous quantitation of fusion protein levels. It remains unclear...proteins. Our recent publication illustrated the quantitative increase in fluorescent signal achieved when...our guest bloggers Erik L. Snapp and Lindsey M. Costantini! Erik Lee Snapp received his PhD in Molecular...

...- a primer for beginners Adenoviral delivery of CRISPR/Cas9 5 tips for troubleshooting viral transductions...formation assay on A549 cells . Alternatively, antibody-based assays are commercially available for faster...ratio for the purified virus should be ~1.3 Quantitative PCR: the number of viral DNA packaged in virions...virions is determined by a standard curve of known quantity and primers specific for a viral DNA sequence....

...packaging transfections Find tips for a first time CRISPR user Resources on Addgene.org Learn how Addgene...titration by qPCR Overview: AAV titering by qPCR quantifies the number of genome-containing viral particles... primer/probe set can be used and samples are quantified by comparing to an AAV standard curve, which ...Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived...

...page Find synthetic biology plant plasmids Find CRISPR plant plasmids ...track, we switched to luciferases to more easily quantify the effect of the different treatments on transformation...transformation success. Luciferases are great quantitative reporters and use a substrate to produce bioluminescence..., Marc-André, et al. "Transient expression of antibodies in plants using syringe agroinfiltration." Recombinant...

... References 1. Ma, Hanhui, et al. “Multicolor CRISPR labeling of chromosomal loci in human cells.” Proc...advantages over their amino acid-based counterparts, antibodies and enzymes, such as: Design and selection ...commercial synthesis (those of us who have used antibodies for diagnostics know how frustrating it can be...hybridization (FISH) where fluorophore-linked antibodies are used to visualize RNA. Low signal-to-noise...

...for transfer plasmids with small inserts (such as CRISPR guides) while a single 72h harvest is best for ...generally a good place to start. If you don’t anticipate that you will need large volumes of lentivirus...

...Addgene blog Engineering the plant genome using CRISPR/Cas9 Using pSiM24 for simplifying plant genetic...the tDNA insertions (usually via seed growth on antibiotic-containing media) A similar process occurs ... the absence of selection. For a protocol for antibiotic selection, check out Harrison et al., 2006. Tips...

...others at the Sainsbury are working on a lot of CRISPR tools for editing plant genomes. We will definitely...using the MoClo system. The paper also gives quantitative data for regulatory elements, which has not ...

...revolution in genome editing methods (most notably CRISPR/Cas mediated HDR) enables the tagging of endogenous...Organized Smooth Endoplasmic Reticulum (OSER) assay (Costantini et al., 2012) measures homodimerization of FPs...the appearance of OSER whorls. This assay is quantitative and is currently the standard for assessing ...: 30783202. PubMed Central PMCID: PMC6381139. Costantini, Lindsey M., et al. "Assessing the tendency of...PMCID: PMC3324619. Cranfill, Paula J., et al. "Quantitative assessment of fluorescent proteins." Nature ...

... interactions Learn how biotin can be used with CRISPR Additional resources Idtdna educational pages... and antibody labelling. In these experiments, biotin is added to a primary or secondary antibody that...that targets your protein of interest or primary antibody respectively. Avidin bound to a reporter is then... on this complex can bind to your biotinylated antibody. This creates a large meshwork of biotin-avidin...

... instance, the popular genome-engineering tool, CRISPR, may one day make techniques used to generate transgenic...from their evolution. Southern blotting and quantitative real-time PCR Southern blotting, named for its...commonly used. In all cases, the result can be quantified by the strength of the signal in comparison to...experimental questions. Real-time PCR detects and quantifies a gene or DNA sequence of interest by recording...Frank Speleman, and Jo Vandesompele. "Real-time quantitative PCR as an alternative to Southern blot or fluorescence...

...genomic loci in living human cells by an optimized CRISPR/Cas system." Cell 155.7 (2013): 1479-1491. PubMed...N-terminus, but this hardly reduces its size. The substantial size of GFP may interfere with the activity or...: PMC5385933. 6. Cranfill, Paula J., et al. "Quantitative assessment of fluorescent proteins." Nature ...PubMed Central PMCID: PMC3918502.  14. Chernov, Konstantin G., et al. "Near-infrared fluorescent proteins...

...CRISPR-Cas9 genome editing may be the hot new way to manipulate gene expression, but other gene manipulation...sequences are highly homologous, preventing design of antibodies specific to each fluorophore. Third, Brainbow...mKate2.) They then successfully generated custom antibodies to each of these proteins and confirmed that ...

...Knockin Plasmids to Disrupt Genes in Mammalian Cells CRISPR Methods for Bacterial Genome Engineering Resources... system to replace a gene of interest with an antibiotic resistance cassette.   Substrate DNA Design..., including selectable DNA fragments, such as antibiotic resistance genes, as well as non-selectable DNA...lambda red expression is that it doesn’t require antibiotic selection to maintain expression of the recombineering...the cI857 repressor to regulate expression. An antibiotic can be used to select for positive clones but...Selection and confirmation of recombinant clones If a antibiotic resistance gene has been inserted, recombinants...recombinants can first be selected via antibiotic resistance, but all clones should be further tested to confirm...

... of other technologies such as optogenetics and CRISPR/Cas9 will help immunologists create even better...with the engineering of fluorescently labelled antibodies made it possible to track cells from one organ...harvesting lymph nodes at different times after antigen challenge, scientists were able to track where ...diverse tissue environments. These studies provide quantitative measurements of cellular motility, interactions...By specifically labeling T cells, B cells, and antigen presenting cells, scientists have been able to ...the bloodstream to invade lymph nodes and scan antigen presenting cells, but two-photon microscopy enabled...scanning was and how fast T cells could jump from one antigen presenting cell to another. Indeed, we now know...

...electrical activity to gene expression, and even CRISPR based gene editing. But, how do you decide which...expression. Experimental output is measured and quantified. Created with BioRender.com.   Optogenetic...optogenetics or biosensors in one system. There can be substantial overlap in the activation curves (Marshel et ...

...vectors fantastic options for delivering shRNA, CRISPR/Cas9 components, and fluorescent sensors. Retroviral... method detects the expression of viral capsid antigen p24, the FACS dosing method correlates viral titre...reverse transcriptase activity assay uses qPCR to quantify viral RNA. All of these methods have been described...

...detect endogenous tagged genes generated through CRISPR/Cas9 knock-in. Finally, NLuc is very inviting for...of NLuc has caught the attention of researchers wanting to perform whole-animal imaging, however NLuc’s...

...sequences cloned into it. gRNAs are used for directing CRISPR/Cas9 to a targeted genomic location, while shRNAs... grown using standard conditions (37 °C, LB, antibiotics, etc.) and stored as glycerol stocks We will...