...then eluted using a high imidazole buffer that outcompetes the interaction between the protein of interest...empirically determined. In addition, some metal ion substrates, such as those containing Co2+, have a lower ...affinity for polyhistidine than more commonly used substrates such as Ni2+ and are therefore more selective... systems, consider using these more selective substrates along with stringent wash conditions.   Find ...

...those sites can be excised by intentional introduction of the Cre recombinase. This is a time-tested system... yet permanent gene excision. Essentially, LoxP sites are knocked in flanking a gene of interest (or a...system but does require knock-in of the LoxP sites, which is more difficult than a simple knock out. Looking...

... form a clean DNA break, Cas3 locates its target sequence and initiates single stranded DNA degradation...exist with unique cutting features and evolved PAM sites, making Cas9 an adaptable choice for many genome...Cas12’s primary difference from Cas9 is that it generates staggered breaks, also known as sticky ends. Cas12a...

...science and engineering. “Fix the Institutions” promotes gender equality in careers through structural ...“Fix the Knowledge” or “gendered innovations” stimulates excellence in science and technology by integrating...combinations of donor/recipient interaction should be tested before being ruled out; there are many factors ...the importance of analyzing sex, the National Institutes of Health (NIH) now requires that sex be included...

...repeat region on its 3’ end. This repeat region associates with the trans-activating crRNA (tracrRNA) which...at the same site, is 2’-fluoro (2’-F), which substitutes a fluorine for the hydroxyl group at the 2’ position...coupling several of them together, for example phosphorothioates with 2’-O-methyl modifications, proved more...Addgene’s validated gRNA repository to find pre-tested gRNA sequences for your next CRISPR experiment....

...allowing an influx of ions into the neuron which generates an action potential (Figure 1). This light-induced...channelrhodopsins to the brain. This delivery route concentrates lots of channelrhodopsins in one region of the...broad transduction of the nervous system which eliminates the need for intracranial injections. When packaged...dopamine is an important neurotransmitter that promotes reward-motivated behavior, if the channelrhodopsins...

...DNA-protein interactions or to analyze chromatin states (CHIP-seq, FAIRE-seq, Cut & Run) and to detect ...signal at the same time point. E. coli’s BirA* was tested at its optimum 18 hour time point, but one minute...physiological expression levels; however, knock-in of BoxB sites into endogenous DNA sequences could allow one to...Human genetic disease. RaPID can also be used to test how RNA variants associated with human disease affect...

...your search and sorting the results (relevance, dates etc). The links for the titles can generally take...you provide, Google Scholar will send you daily updates with relevant literature. Organizing the literature...margins. If you prefer, printing and hand-writing notes is still possible, but not feasible for every paper... University. She is a fan of photosynthesis and writes about autotrophs and biochemistry on her blog. ...

...requires the much less efficient double stranded templates, rather than the more efficient single-stranded... development, regeneration, and certain disease states. However, it’s difficult to observe the cell cycle... from Eastman et al., 2020. The researchers tested several scenarios from embryo development to myeloid...paralogs. The exon-deletion hybrid gRNA library deletes human alternative cassette exons and disrupts genes...

... into the city of Guangzhou and see some of the sites. Most fascinating to me was the outdoor market where...tuned for big developments in this field. Nobel Laureates and the ever expanding importance of RNA The second...of Georgia studies the CRISPR/Cas system as it relates to its natural role in prokaryotic immunity to ...phage vectors with CRISPR/cas9 sensitizing gene cassettes. Thse vectors are engineered to replace the drug...

... helps to maintain a consistent stream of light. Test your implant by connecting it to a patch cable connected...stimulation only under certain contexts/locations in the test apparatus and thus individual laser stimulation ...channel, Channelrhodopsin 2 (ChR2), exists in several states: closed/light receptive, open, closed/light insensitive...length for your specific behavioral apparatus or test but not so excessively long that it becomes tangled...

...through 3 major steps to select for optimal sybody candidates. Figure 2: Outline of the process for selection...display mRNA from the enriched pool of sybody candidates are then reverse transcribed into cDNA fragments...cloning technique which uses Type IIS restriction sites (Geertsma, 2013). Once the phagemids are constructed... narrowed-down pool (usually 10-30) of sybody candidates are sequenced for identification. All unique ...

...reach zooxanthellae, the coral’s symbiotic dinoflagellates that are photosynthetic (Salih et al., 2000...substrate, chromoproteins don’t require other substrates for screening. Thus chromoproteins can be used...common chromoproteins used in the lab come from eukaryotes, they can be difficult to directly use in bacteria...plasmid carrying the desired genetic modification integrates into the genome (Figure 3). Here, the chromoprotein...

...fluorescent signaling in the presence of dsDNA. Image courtesy of: Ygonaar    Taqman is more sensitive than...of the Taqman reaction can be seen to the left, courtesy of UPODMG 1516 irivbrro ddPCR  Droplet digital...see image, left, of droplet immersion technique, courtesy of CAR15.) Each droplet contains a primer and ... of reactions, it does not need to be run in triplicates. ddPCR is an extremely accurate application, ...

...addition of a probe based fluorescent dye that intercalates with any dsDNA and the use of a fluorometer ...concentration, unlike qPCR where results can vary across replicates. ddPCR can be used to quantify DNA sequences ...primer sets.  Multiple template PCR - multiple templates with different primer pairs that align to the ...quantify the concentrations of multiple starting DNA templates in one sample. It is important to note however...

...digest (the ITRs contain SmaI and XmaI restriction sites). Another way is to transform AAV transfer plasmids...release the virus particles from the cells. Cell lysates are then treated with benzonase immediately before... bit of flexibility since I can store the cell lysates at -80 °C for up to 6 months before purifying the...the physical titer generated by qPCR is useful, I test a few different concentrations of virus, or multiplicity...

... structure of a protein, based on its atomic coordinates. These wire models were useful in that they not...download a pdb file of the structure’s atomic coordinates, and “open” these structures with molecular viewing...then activate two different endonuclease active sites. Fortunately, there is never just one model of a...gymnastics that this protein must undergo as it interrogates your genome. If you are interested in experiencing...

...protospacer. The resulting double strand break (DSB) stimulates DNA repair mechanisms that can be exploited to...rules that result in a decrease in available target sites. Regulating gene expression with truncated gRNAs...Altering the length of gRNAs targeting different sites of interest thus represents a straightforward means...transcriptional activation or repression at the respective sites.   Many thanks to our guest bloggers Alissa Lance-Byrne...

...becomes impossible (or at least unreasonable) to test them all. This is where PRIDICT comes in! Evaluate...editing efficiencies in high-throughput libraries My labmates and I wanted to find a way to predict the efficiencies...select some of the best designs (e.g., top 5) and test them in their labs. Figure 2: Guidelines...most promising pegRNA designs and especially facilitates the use of prime editing in pooled settings with...

...using much smaller regions of homology, which facilitates easier cloning. An overview of PITCh. ... 5' and 3' microhomology and PITCh-gRNA binding sites. This PITCh plasmic is cotransfected with a plasmid...PITCh-gRNA, and the locus-specifc gRNA. The PITCh-gRNA creates two DSBs in the PITCh plasmid to release the cassette... by CRISPR design tools at crispr.mit.edu. They tested their system in HEK293 cells, integrating the GFP-Puro...