... 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...

...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 ...

...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...

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

...delivered: fluorescent proteins, optogenetic sensors, CRISPR elements, etc... *These are only starting recommendations...concurrently to minimize the impact of neutralizing antibodies developed in response to the first exposure to...AAV-PHP.eB. For this reason, the Gradinaru lab anticipates that high-dose AAV-PHP.AX will be the capsid ...efficiency, and is not neutralized by pre-existing antibodies against AAV9-based vectors (and vice versa), ...

...Experiments Experiment  Detection Method Notes Quantification of a process Plate-based assays (plate reader... HPLC (proteins or other molecules) Isolate or quantify cells, proteins or other molecules that have been...Autophagy Sensor Read about Aptamer Fluorophores Use CRISPRainbow for Genome Visualization Resources on Addgene.org...