...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...

...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...

...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...

...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 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...

...may include reduced saturation density, decreased rates of growth, or clumping. To ensure the best possible... culture-based experiment, labs should routinely test for mycoplasma contamination. There are several ...with freely available positive controls. Routinely test your cell line for mycoplasma and discard it if ...routinely produce lentivirus may want to consider testing a variety of cation and serum sources to ensure...

...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 ...

...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...

... (pLemo) Rhamnose-tunable T7 RNAP expression alleviates inclusion body formation. Expression vector cannot... recA+, KmR Expression of toxic proteins  * Denotes the presence of an additional plasmid-- make sure...setting up a small-scale expression experiment to test variables such as temperature, time, and media conditions...temperature or moving it to a bench-top shaker 10-20 minutes before adding the inducer. Growth Media: Changing...

...tightly regulated. Common promoters for eukaryotes and prokaryotes We have included two reference tables...bacteria this is done by a single enzyme; however, eukaryotes have muliple polymerases which are each responsible...expression mRNA Yeast promoter containing Gal4 binding sites, commonly used in Drosophila Specific Requires the...

...approach is most well suited, since it routinely generates knockin efficiencies of 1-40% with G418-selected... AAV to deliver their repair template to mouse zygotes and thereby correct a mutation that causes Hemophilia...technique to efficiently deliver large repair templates for applications that require large gene fusions...Porteus. "CRISPR-mediated integration of large gene cassettes using AAV donor vectors." Cell reports 20.3 (2017...

...different splice site donor and acceptor sequences were tested and with the optimal sequences, the group achieved...homologous recombination approach delivers large donor templates using two sequential homology directed repairs...2017) CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors. Cell Reports 20:750–756...PA (2002) An mRNA Surveillance Mechanism That Eliminates Transcripts Lacking Termination Codons. Science...

..., followed by an intramolecular reaction that generates the chromophore (Tsien, 1998). Importantly, this...absorb light at 488 nm. The ratio between the two states is reflected by the pKa, which is around 6.0 for...relatively small, adding GFP may alter diffusion rates. Smaller genetically encoded probes are based on...Adjobo-Hermans, 2011). Structural information facilitates the choice of the proper site, although it still...

... Fig 1: Quote from Michael Ende, courtesy of morefamousquotes.com A good listener absorbs and remembers...college, when I discovered that many of my old suitemates stopped talking to each other if I was not there... diagram of the Thomas-Kilmann Conflict Model, courtesy of https://managementweekly.org/thomas-kilmann-conflict-resolution-model...

...molecules, knowing that where you see fluorescence indicates your target’s location. Some other terms you might... The biggest difference between IF and ICC/IHC relates to the type of secondary antibody you use, so as...corresponding protocols. Other researchers may also have tested an antibody in IF and will share their findings...of interest. Thankfully, there are a number of websites compiling antibody data that can help you compare...

...-target sites with up to 7 mismatches to the gRNA target sequence. Kaminski et al. next tested their procedure...targeting of the enhancer region near the NF-KB binding sites increased activation of HIV promoter-luciferase ...expression of Cas9/gRNAs, which could result in higher rates of off-target cleavage. In both cases, translating...

...not require the presence of specific restriction sites (or lack thereof) nor a serious time commitment....of fragments, but uses site specific restriction sites (Type IIS restriction endonucleases) to cleave DNA...that the vectors and DNA fragments contain these sites at the correct location and NOT in the middle of...flanked by orthogonal versions of the attB and attP sites recognized by the integrase. This method requires...