...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...

...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...

...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...

...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...

...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...

...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...

...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...

... 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...

...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,...

... Miller, founder and principal of J. Miller & Associates, Inc., a national grant consulting firm, and ...Professional Certification Institute (GPCI). GPCI evaluates a candidate's expertise (a minimum of 3 years ...CSMS is the founder and principal of J. Miller & Associates, Inc., a national grant consulting firm, and ...and evaluation. She and her team at J. Miller & Associates have secured more than $70 million dollars in...

...spontaneously and without additional co-factors, substrates, or enzymatic activity – it only requires the...means to many functional ends. We’ve listed our favorites below, but many other uses currently exist, and...used; one fused to each protein or domain being tested. Find FRET plasmids here. Split EGFP: An alternative...(FACS): This is a type of flow cytometry that separates mixtures of cells into distinct populations based...

... Cas13a binds and cleaves RNA rather than DNA substrates. In terms of structure, Cas13a shares no homology...molecule through a uracil-rich stem loop and facilitates target binding and cleavage through a series ...changes in the Cas13a molecule. Like Cas9, Cas13a tolerates single mismatches between the crRNA and target...PMC4660269. 7. Joung J, Ladha A, et al., ‘Point-of-care testing for COVID-19 using SHERLOCK diagnostics.’ medRxiv...

...modification sites and even affect protein function (Mauro and Chappell, 2014). Different rates of translation...base-pairing mechanism for translation initiation in eukaryotes. Nat Struct Mol Biol 13:30–34 . https://doi.org...Zavolan M (2013) Analysis of CDS-located miRNA target sites suggests that they can effectively inhibit translation...

...labeling. For labeling the plasma membrane, they tested different target sequences for improved selectivity... Umemura have described a simple method which alleviates the inclusion body problem and makes nanobody...and gRNAs. The multi-gRNA piggyBac vector integrates into genomic DNA with the help of the piggyBa... Image from Hazelbaker et al., 2020. The lab tested this system by repressing and activating expression...

...the immunoglobulin, leaving the antigen binding sites free to capture the target protein. The beads can...indiscriminately, causing competition for binding sites. In cases like these, antibodies can be directly...sample. It is important to remember that sample lysates will also contain proteases and phosphatases that...sample.  Eluting the target protein for downstream testing Once the non-specific proteins are removed through...

...as well as their bin-mates.  They liked us. They really liked us.  The testimonials from participants in...explained my situation and asked if she had a few minutes to point me in the right direction for my future....” These sentiments were echoed in the other testimonies we gathered. The program definitely caused a ...

...The data is also used to determine recombination rates in FLEX/DIO/fDIO constructs. Endotoxin Endotoxin...sterility. Transducibility Some viral vectors are tested in vitro and in vivo for gene expression and/or...obtained directly from Takara, and is routinely tested for mycoplasma contamination using mycoplasma detection...two weeks post-thaw, cell culture supernatant is tested for mycoplasma contamination. To date, Addgene ...