...found that the accuracy of ICE analysis results was highly comparable (within r2=0.96) to that of NGS data...coefficient (r) is also computed and reported.  The higher the R2 value, the more confident you can be in ...the Download Analysis Data button on the bottom right of the analysis screen. ICE analysis details Contributions...genomes that contain the indel. The discordance plot (right panel) shows the level of disagreement between the...that continues after the cut site, representing a high level of sequence difference. Traces The Traces...

...SDS-PAGE...Electron microscopy… Any guesses? That's right... ways to titer AAV! The point is there are many...linear region of the standard curve. In addition, high and low-concentration samples can also behave differently... reference sample of known titer, which is at a higher titer than the AAV RSM. By using these samples ...) AAV titers are approximations and they depend highly on the experimental conditions used, titer values...PMID: 10455443. 3. Hindson, Benjamin J., et al. "High-throughput droplet digital PCR system for absolute...

... procedure (Mortensen et al., 2019). Our method might provide some useful guidelines for the development...transformation was successful at all. Once we were on the right track, we switched to luciferases to more easily...simple and easy technique to implement. Success might vary between experimenters. Vacuum infiltration ...especially problematic with reporter genes as you might observe expression from Agrobacterium, but not the...leaf transformations, but we hope that these tips might help to get you started. Once you have established...

... develop and optimize the method termed Specific High Sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK... the promiscuous cleavage and degradation of neighboring ssRNA and ssDNA by Cas13 and Cas12a, respectively... of CRISPR-based diagnostics. SHERLOCK- Specific High Sensitivity Enzymatic Reporter UnLOCKING Cas13 (...SHERLOCKv2 provides a method to detect nucleic acids with high sensitivity and specificity without compromising...the ability to rapidly detect nucleic acids with high selectivity and sensitivity from patient samples...

...scientists achieve a high titer (reviewed in Joglekar and Sandoval, 2017). This high titer is one of the...your virus, there are many other reasons when you might pseudotype your virus:  Lowering cytotoxicity. ...reaching the target cells. VSV-G is also toxic at high concentrations as described above and is inactivated...vector to be transported retrograde and also has higher neuronal transduction efficiency and a better safety...

... This powerful negative selection system ensures high editing efficiency; the only non-edited cells to...The simpler system of pCRISPomyces-2 displays a higher editing efficiency, perhaps due to its condensed...Streptococcus thermophilus, Cas9sth1 as a robust and highly active enzyme for transcriptional silencing Mycobacterium...efficiency of knockdown and developed new, more tightly controlled, Tet responsive promoters to avoid leaky...dCas9 variant as well. Furthermore, they provide a high-fidelity (HF) variant of the BE3 that further improves.../10.1038/nbt.3011 Cobb RE, Wang Y, Zhao H (2014) High-Efficiency Multiplex Genome Editing of Streptomyces...

...change and screen a few hundred clones to find the right one Step 2 – Edit the cells a second time using ...corrects the blocking mutation and again identify the right clone from a few hundred. From these two steps, ... few hundred clones - Still better than 10.000, right? (Kwart, Paquet, et al  2017). Find Additional CRISPR...increasing mutation incorporation efficiency, we might exploit this relationship to direct the zygosity...Additional Resources on the Addgene Blog Learn about High Fidelity Cas9 Variants How to Design Your gRNA Delivering...

...created vectors for Lambda Red that contained one of eight bacterial toxin genes isolated from E. coli or Psuedomonas...both rhamnose- and arabinose-inducible promoters. Tight control of toxin expression is absolutely necessary...induction system is well-characterized and quite tight (Giacalone et al., 2006). The Chen lab demonstrated... selected the three toxins so that at least one might be useful in a broad array of pathogenic Proteobacteria...expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system." Biotechniques...

... a great plan to start consistently knocking out high-quality videos for you all. The next day...we got...Everything we had set up involved multiple people in tight locations. Conference rooms for on-screen presenters...raised on an ironing board, giving it the perfect height (Fig. 2). We recorded video using a simple, external...comfortable on camera while thinking about things like lighting, camera angle, and shot composition.  So back ...could transmit a signal between a wall. There was a SLIGHT delay, but I could work with that.  Focus: Next...

... cell mixes, like bulk lympho-/splenocytes, you might only be interested in one of the various cell populations...markers of all cell types except CD8+ T cells. We might choose the markers CD4, CD19, and CD11c, which would...rest can be excluded, or “dumped”. From left to right: B cell, CD4+ T cell, dendritic cell, CD8+ T cell...host species reactivity. When sorting cells with a high resolution for specific downstream applications,...immunity. When he's not in the lab, Paul enjoys weight lifting, sailing, and reading.   More resources...

...cells. However, for some applications, integration might be considered a double-edged sword with unintended... lentiviral vectors, how they work, and why you might choose them over integrating lentiviral vectors....lentiviral vectors? There are several reasons why you might choose an IDLV over a lentiviral vector: 1. No risk...mind is that titering these IDLVs may require a slightly different protocol than regular lentiviral vectors...Reyon, D., Joung, J. K., & Sander, J. D. (2013). High-frequency off-target mutagenesis induced by CRISPR-Cas...

... together on a tight budget. To help popularize our kit resources,  we’re highlighting just a few of ...covering a wide range of fields from Addgene. What you might not know is that Addgene distributes curated collections...Morgan’s blog post on the PUF Assembly Kit for some insights on its potential from its creators. We hope you...

...amount of high-quality DNA, real-time PCR has several advantages including easier automation, higher throughput...method called pyrosequencing. The drive for faster, higher throughput and less expensive sequencing fuels ...commercially available although they are reported to have high error rates (10). It is projected that nanopore-...and research progress. These will bring faster, higher throughput, and potentially more accurate techniques...

...untreated individuals. Symptoms of AIDS include rapid weight loss and an increased risk of infection, including...models, and it’s difficult to tell if one approach might be more successful. “Shock and kill” has the advantage...expression of Cas9/gRNAs, which could result in higher rates of off-target cleavage. In both cases, translating...each of these gRNAs will need to be validated for high on-target binding and low off-target binding. It...stable expression of Cas9/gRNA. Viral production is higher in this model than in latent HIV-1 infection, which...

...the perfect empty backbone, made sure to add the right tags to your gene, and may have even put a fluorescent...are marketed for a specific purpose: fast growth, high-throughput cloning, routine cloning, cloning unstable...  Propagation of ccdB-containing plasmids Hte   High transformation efficiency deoR constitutive expression...synthesis Allows uptake of large plasmids hee   High electroporation efficiency supE44 (glnV44)   Suppression...+)  XL10 Gold Tetracycline and Chloramphenicol High competency cloning and propagation of large plasmids...

...This Depositor Week, we're highlighting eight amazing posts on research done in our depositor labs. Some... Andrew Willoughby introduced us to RUBY-red, a bright red pigment reporter for plants.  Cas7-11 and PASTE...

...community like Watertown to bulk purchase energy from a higher percentage of renewable sources. This program offers...sources and we have chosen to participate at the highest level offered. As a result, we will be drawing ... our electricity from renewable energy projects right here in New England- the majority of which are solar...temperatures, biosafety cabinet ventilation, and lab lighting/HVAC requires a significant energy expenditure...

...Sleigh bells, please! It's time to announce the winners of the 2022 #DeckTheLab contest!  and it's.....in our building. Unfortunately, snow is thawing right now, so today noone could see my (not so) little...                     A post shared by 草方 (@gracie_fang) That’s right, researchers of the world. Cap your Eppendorf tubes...Instagram! Happy decorating, all, and to all a good light!    🤩 our lab in Bayliss Building @SMS_UWA is getting...

...vector pAAV-CAG-RdLight1 AAV9 Biosensor Tian New viral vector pAAV-hSynapsin1-RdLight1 AAV9 Biosensor...lab. Also of interest is the Tian lab's pAAV_hSynapsin_psychLight2 (AAV9), which contains a biosensor that...Biosensor Tian New viral vector pAAV_hSynapsin_psychLight2 AAV9 Biosensor Tian New viral vector pAAV.hSynapsin.SF-iGluSnFR.A184S...

...that infectious wild-type viruses were produced in high-titer retroviral stocks generated by the transfer...In this kind of approach, a population of cells might express only the tTA or your transgene resulting...developed, but these vectors are quite large and you might get lower viral titer when using them.   Regarding.... If you are transducing non-adherent cells you might need to centrifuge your cells with the virus in ...Trouble Shooting Viral Transductions Learn Why You Might Want to Use AAV in Your Research Lentiviral Vector...

...unless the source of culture is irreplaceable or highly expensive). Detecting mycoplasma contamination ...induces a noticeable color change. This method has high sensitivity but low specificity (Degeling, 2012)...required in this process because result interpretation might be difficult. The low density of mycoplasmas, contamination...media as much as possible and the ones that are tightly packed with seals. Use disposable pipettes to avoid...exposure of the cell culture to air and make sure to tighten the flask cap before placing them in the incubators...

...decades of my life following a very straightforward conveyor belt from high school to college to graduate school...Veterans Health Administration, I could never get one straight answer about what science policy even is! In this... “little p” policy.  “Big P” policy is what you might typically think of. It occurs in a government setting... challenging if you are trying to apply for an outright job in a field where you have no specific scientific...

...and four chaperones from Denis-Yarmouth Regional High School visited our labs in Cambridge, MA to learn... Outreach Scientist Jessica Welch showed some highlights of his travels from 2015, including a great European...Website Learn more about Dennis-Yarmouth Regional High School Resources at Addgene  Read more Inside ...

...working with AAV or lentivirus in your research, you might want to use one of our virus backgrounds. Addgene...backgrounds for you as well! If you use FRET, you might be interested in our fluorescent protein background....           Did you get the joke? If not, you might want to check out Addgene’s Fluorescent Proteins...background.     We hope these science backgrounds will brighten up your virtual meetings!  ...

...It’s well known that exercise can help fight stress and depression, and now researchers have reported...including unpredictable loud noises and flashing lights - that sent average, control mice into depression...know about you, but I think I could use a good run right about now. And, if you are looking to get your hands...for studies in your own lab, you’ve come to the right place. Get started with PGC-1 plasmids from Addgene...

...Directors brings human resources experience and insight to the now 13 member board at Addgene. Let’s meet...hope Addgene will accomplish in the near future? Right now it’s a matter of maneuvering through the impact...volunteer appointments included the Belmont Human Rights Commission and mediator for the Massachusetts small... dedicated to volunteerism and ran the Mayor’s Midnight Sun Marathon in Anchorage, for the Leukemia Society...

...to the field. Fortunately, our bodies have been fighting cancer for far longer than we’ve been studying...natural abilities, we’re gaining new ground in the fight against cancer in its many forms. The dichotomous...cancer stops growing, and the immune system stops fighting. Such quiescent cancer may rear its head once ...infection can further divert the immune system from fighting cancer. Surgical removal causes increases in tumor... a sequence of immune reactions that aid in the fight against cancer. Sipuleucel-T by the Dendreon Corporation...

...instance, you might use it as part of a flex switch you’d like to work with in neurons or you might use it in...Cre-controlled gene of interest makes pmSyn-BFP-Cre highly modular. Further uses of pmSyn1-EBFP-Cre Of course...brain. There are many possibilities enabled by this highly modular AAV - that’s probably why it’s so popular...

...bigly. But such a project should not be undertaken lightly. Whether executed in arrayed or pooled format there...are just as critical for success. Sometimes, you might have a few positive control genes on which to test...execute genome-wide perturbations. So, while some might argue that the only way to do something is to do...still offering the potential for true biological insight. Pooled and individual plasmid formats A first ...

...group? The Ellis Lab self-organizes cinema nights, game nights, and trips to eat out together. “We also ... celebrate your lab’s achievements? Tom Ellis highlights the importance of acknowledging his teams successes...

...? If so, you might want to find additional sources of wisdom. While somewhat lighthearted, the article...article does highlight key characteristics of a good mentor: Yoda image courtesy of jorenl/Vecteezy.com...

...share? And how they are connected? Here are the highlights written as 8 tweetable facts. Note: The images...most Twitter users. Biologists rank #8. Figure 1: Highly Represented Scientific Disciplines on Twitter. ...disciplines: social (left), life (center), and physical (right). How do you use Twitter as a scientist? If you ...

...them share their reagents, we knew our role in fighting the pandemic could be even more impactful by reaching...scientists working in a for-profit setting.  In light of the need for basic research during the current...University of Iowa University of Minnesota See highlights from the COVID-19 collection Partnership with...

...Agrobacterium, along with a selectable marker. Left and right T-DNA borders specify the region of DNA that will...codon-optimizing the Agrobacterium ORI, they achieved much higher DNA yield and transformation efficiency in both...that gene expression from the M24 promoter is much higher than that from the commonly used 35S promoter. ...transfer. The M24 promoter provides constitutive, high-level gene expression and is functional in a variety...

...been in space is pretty small). Therefore, I was delighted to be invited to the ceremony for surely what ...non-scientist from Bloomberg news. Given the topic it might have been better to have a scientist up there, but... viruses to give information to the computer to fight the known viruses. Bacteria have figured out how... virus sequence to enable them to recognize and fight it forever. He emphasized the potential of this ...

...research". The family entered the biomedical field’s spotlight after researchers discovered heavy-chain only ...reading frame Ability to access unique epitopes with high affinity, due to a greater ability to target concave...cellular functions, like in this early example we highlighted back in 2015, where sdAbs were used to create...

...toxic to both lines, it probably has either high toxicity or high off target effects, both of which make it... druggable target). Diseases with defined, straightforward causes (a specific mutation in a known gene...targets by CRISPR can accelerate the drug pipeline. High-throughput CRISPR screens can be used to inhibit...

...Gates’ Foundation is involved with was one of the highlights of the conference. It was inspiring to see how...trusty spray bottles! We also raffled off one of our highly sought-after "nice genes…" tshirts, and the lucky...Fluorescence Labeling Plasmids from the Barbier lab). Bright and early on Saturday morning, John Doench’s opening...

...skills to get the work done efficiently and with high quality. Their work involves coordination and can...t mean going rogue, pointing out problems or highlighting criticisms. To be given the opportunity to take... by Peter Fuda and Richard Badham Leadership Insights for Engineers, Scientists and Computer Professionals...

...planning to transform right away OR you can store the reaction at -20C overnight. 4. Transform TOPO Cloning...and requires no post-PCR procedures. Sounds easy right? The technique relies on the basic ability of complementary...efficiency).  5. Select and Analyze 10 White or Light Blue Colonies: You can confirm the presence of your...