...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...

... Viruses are intracellular parasites and natural vehicles for genetic information. Therefore they make...Addgene: Follow our viral vector blog posts for updates on the most recent deposits, protocols, and tips...selection. One of the scientists who created pBabe writes how its name came about here. Cloning and expressing...MuLE (Multiple Lentiviral Expression) system facilitates the simultaneous introduction of multiple genetic...

... 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...

...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...

...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...

...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...

...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...

...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, ...

...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 ...

...CRISPR-mediated purification of a specific genomic region alleviates these issues as a region of interest can be targeted... relax imprinting (Zhang et al., 2017). This upregulates expression of IGF2, a characteristic of many ...analysis of bacterial genome functions." BMC research notes 11.1 (2018): 387. PubMed PMID: 29898790. PubMed ...modular RNA-guided regulation of transcription in eukaryotes." Cell 154.2 (2013): 442-451. PubMed PMID: 23849981...

...insert either randomly, or at fixed genomic target sites.  This obstacle was recently surpassed with the ... bound by QCascade (Fig. 1B). The process also creates a 5-bp target-site duplication (TSD), a hallmark...insertions and intermediate structures known as cointegrates (Vo et al., 2020; Strecker et al., 2020).  CRISPR-transposons...to multi-copy genomic integration of metabolic cassettes (Zhang et al. 2020); however, the technology potential...

...tumeficans - a plant pathogenic species that stably integrates transfer DNA (tDNA) into the genomes of the plants...of great options, but here are a couple of my favorites. Plasmids from the Tsuyoshi Nakagawa lab are binary...difficult. Don’t leave your plants on selective plates for too long: even resistant plants will be affected... the National Academy of Sciences of the United States of America. National Academy of Sciences, 112(18...

...recombination reported in all laboratory E. coli strains tested so far. The use of this pathway as a cloning tool...for example for subcloning genes from different templates. All PCRs are run as an 18 cycle, 25 μl single-tube...We use XL-10 Gold E. coli, but have successfully tested DH5a, TOP10, and Mach1. No common lab cloning strain... the whole vector sequence between modification sites (see Figure 3), producing multiple fragments with...

...of mouse zygotes, through a process known as CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) (Chen et...electroporation (A in the figure above), which generates pores in the cell membrane, allowing for entry...by CRISPR ribonucleoprotein electroporation of zygotes." Journal of Biological Chemistry (2016): jbc-M116...

...time and effort to test and optimize our vector - overall, I spent two years testing and reformulating ...H134R) in A2 norepinephrine expressing neurons. We tested four serotypes containing an expression cassette... of that data was published.    Fig. 1: Testing of three different AAV serotypes (AAV1, 8, 9) injected...

... gene. In this special CREATE cap plasmid, loxP sites flank the downstream polyA sequence. When viruses... in the Cre-expressing tissues. This inversion creates a template that’s suitable for PCR amplification...which, in the case of Chan et al, was neurons and astrocytes. Why screen for IV deliverable AAVs? Typically...tracing, aka determining where a neuron’s axons and dendrites extend to. The keys to this method are 1) high...

...such experiments, small-molecule inhibitors are tested in parallel for their ability to prevent BiFC resulting...preferably, some information about the structures of the tested PIPs. Many split FPs have been validated for BiFC...- and C-terminal end of each PIP and empirically test which combination provides the best fluorescent ...appropriate controls may requires that you empirically test a number of mutants to identify those that inhibit...

...culture here at Addgene has remained paramount. Candidates who interview at Addgene frequently ask the question... at Executive Director Joanne Kamen’s home- a testament to the familial aspect of Addgene. As our size...time for fun, everyone is confident that their teammates and project collaborators have completed their...these examples we’ve done annually - they are fan favorites! 6. Keep it simple This may be an obvious one,...