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...sections, while still creating scarless plasmid inserts.  Want to Learn More about DNA Assembly? Check...polymerase used in the amplification. This tool allows instant updates as fragments are added, deleted, or reordered...(sgRNA). Using only one single-stranded oligo, instead of the more typical two oligos, is more cost effective...linearized plasmid with a synthetic oligo that contains a region of homology and any desired edits to ...amplified with overlapping regions of homology and inserted into a linearized cassette, ready for transformation...

...in monoallelic diseases In 2013, a team led by Jinsong Li from the Chinese Academy of Sciences found that...independent team led by Hans Clevers at Hubrecht Institute in The Netherlands used the CRISPR/Cas9 genome...handle on how cleavage sites are repaired.” Those insights will be critical for scientists’ ability to influence...addition, the current work in developing anti-CRISPR proteins aid the limitation of these mutations, to control...s Disease.” Journal of Alzheimer's disease & Parkinsonism. https://doi.org/10.4172/2161-0460.1000439 6...

... Biological repositories that distribute mouse strains, plasmids, cell lines, and much more have been ... Bioresources Cell Bank, part of the National Institutes of Biomedical Innovation, Health, and Nutrition...19 plasmids and resources! Case study 3: Mouse strains Reagents developed during the SARS-CoV pandemic...The Jackson Laboratory, a nonprofit research institution, has been able to rapidly propagate this strain... Aliyah Weinstein...

...based on your downstream application, time, and instrument availability. UV absorbance Using UV absorbance...acids (DNA and RNA) absorb maximally at 260 nm. Proteins on the other hand absorb best at 280 nm and organic...you can compare the fluorescence of your sample against this curve to quantify your DNA prep. Though this...this method doesn’t tell you about contaminating proteins or chaotropic salts. Capillary electrophoresis...read with a standard ELISA reader if no other instruments are available. Some last reminders about quantifying...

... when a researcher decides on a new project and begins by trying to reproduce someone else’s result. Then...time that they thought would be moving forward instead trying just to get going. It’s like being stuck... in science needs both a job and funding, so institutions and funders, to a large extent, hold the keys...is clearly room to do more. If we could really instill a culture of rewarding good scientific citizenship...UK and then a postdoc at The Scripps Research Institute in La Jolla, California. She joined Elsevier after...

...second GFP antibody?  Similarities:  Generated against full length GFP and converted to recombinant form...Isotype IgG2a Differences:  Distinct variable domains that recognize different epitopes on GFP Monoclonal...light over the last decade, but the fluorescent proteins (FPs) necessary for many of these applications...consider hfYFP and LSSmGFP.     Fig. 4: hfYFP retains folding and fluorescence in high molarity GdnHCl...Campbell, B.C., et al. Chemically stable fluorescent proteins for advanced microscopy. Nat Methods 19, 1612–...

... may feel it’s overwhelming or challenging. So instead, let’s focus on practicing with a short and accessible...information? This can be physical or digital. For instance, you would present information differently in ...image on a protocol page on our website, as an instructive step-by-step guide.  Finally, when writing your...after centrifugation and the syringe needle is inserted bevel-up at the interface between the 40% and ...captured at the 40% layer. To collect purified AAV, insert a syringe at the interface of the 40% layer containing...

...readouts for levels of different chemicals, hormones, proteins, etc., that you are interested in. The most obvious...could be a microscope, plate reader, or any other instrument which measured GFP levels and converted that ...includes tension monitors associated with specific proteins of interest such as actinin. When tension is high....2019.00474 Resources on Addgene.org Fluorescent Proteins: Biosensors Viral Service: Biosensor AAV Viral...Epigenetics with a FRET Based Biosensor Fluorescent Proteins 101: Fluorescent Biosensors ...

... at the Hotchkiss Brain Institute. He previously worked at The Allen Institute for Neural Dynamics and...tool in neuroscience research. These fluorescent proteins light up in response to calcium, which is more...spectrum (Figure 1). The GCaMP series (e.g., jGCaMP8) remains the gold standard for green fluorescence, while...Additional Resources on addgene.org Fluorescent Proteins: Biosensor Plasmids In-Stock AAV Vectors: Biosensors...

...AAV? A: One potential strategy is to use split-inteins to mediate trans-protein splicing. Note, however...possible to make an efficient AAV prep for a 4.2 kB insert with the CMV promoter using the AAV-BR1 capsid?...bringing total genome length to ~4.7kB.  If your insert alone is 4.2 kB and CMV promoter+enhancer is 0.5...experiments using PHP.eB in the brain give some insight into codelivery efficiency at different doses. ...tropism to other serotypes, but reproducible success remains elusive.  There has also been a lot of effort in...

... thanks to our guest blogger Rachel Rubinstein! Rachel Rubinstein is currently a Field-based Product Manager...post was contributed by guest blogger, Rachel Rubinstein, a field-based software product manager at Oxford...your network to your advantage  As the profession gains more traction in the sciences, I believe that more...customer in mind. You can follow her on twitter @rgrubinstein. Additional resources on the Addgene blog Discover...

...certified health insurance executive (CHIE). He is a member of both the Internal Institute of Auditors and... Directors brings complementary experience and insight to the now 10 member board at Addgene. Let’s meet...at both MetLife & Ernst & Young, LLP, auditing insurance, finance and high-tech industries. He has earned...Addgene blog Learn more about about Addgene in our "Inside Addgene" blog series Subscribe to the Addgene blog...

...comprised of two tandem fluorescent proteins: a relatively pH-insensitive RFP variant, DsRed.T3 and a pH-sensitive...the degradation rate of radio-labeled long-lived proteins as a proxy for autophagy. Cells are cultured with...washout period removes radio-labeled short-lived proteins primarily via proteasome degradation. Autophagy... to giving more fine-grained detail about what proteins or organelles are being degraded) and it is slow...

...resources. There are three types of cognitive load: intrinsic cognitive load is the effort associated with a...show frustration if they're incorrect; take it instead as valuable feedback.  Listen. Think about the ...strategy. Don’t assume it’s the audience’s fault; instead look for ways to improve how you're communicating...If something I was communicating didn’t click, instead of blaming the audience, I would think about how... wasn't. Maybe I needed to employ a visual aid instead of describing something verbally. Sometimes a group...

...for every assay. Antibodies that are generated against a linear antigen tend to work very well for applications...application. Take immunohistochemistry (IHC), for instance. While an antibody may be validated for IHC, not...region highly conserved across species? If the proteins are highly homologous between your target species... each isotype or species to easily discern the proteins in your assay.  Narrowing the field After working...compare the staining patterns of multiple antibodies against the same target; tagged protein strategies that...

...awards to early career scientists using fluorescent proteins in their research. These awards help early career...biosensor fuses MPST to roGFP2. Because roGFP2 contains two cysteines near the chromophore that are susceptible... to better understand the processes that happen inside the cell,” says Pedre Pérez. Figure 2: ...changes following opioid withdrawal, providing insight into new methods for treating patients in recovery...indicator which had improved performance in live mouse brains. Currently, she is using the improved voltage indicator...

...discussions.  Pro tip: Instead of connecting with someone you don’t really know, follow instead of connecting....information available. When I am speaking at a new institution, for example, I will often see a whole bunch ... “a postdoc from this Uni viewed your profile” instead of “Joanne Kamens, PhD viewed your profile.”  The...option for sending LinkedIn messages to Click Send instead of Press Enter to Send. That way you can type a...

...postdoctoral researcher at the Noble Research Institute, co-instructed the ASPB workshop and authored a three-part...well received, and then when the team started brainstorming ideas for the next workshop, they realized that...in the group was able to make it physically to instruct the workshop at the conference, but because each...of the scientific community. Lessons learned Instead of continuously talking about a problem and potential...

...destination vector + insert may spontaneously religate, this transient construct retains functional Type IIS...in the final construct. The destination vector contains sites with complementary overhangs that direct...complete with helpful tips and tricks, see The Sainsbury Lab website or Engler & Marillonet. Golden Gate...technology has adapted Golden Gate cloning for inserting the appropriate oligonucleotides specifying a ...

...use a standard strain, like DH5α, grown at 30 °C instead of 37 °C. If you’re unsure about the integrity ...prefer to store freshly harvested virus at 4 °C instead of storing it briefly at -80 °C. If troubleshooting...option may be to avoid freezing altogether, and instead to use the virus immediately upon harvesting. Interestingly...appear as a white pellet. The pellet can then be rinsed and resuspended in cold sterile PBS.  Alternatively...of ensuring viral production is to perform PCR against components of the viral vector using the harvested...