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... it has been challenging to introduce CRISPRi machinery into diverse bacteria species. Existing protocols... disease-associated. Mobile-CRISPRi: a modular approach for CRISPRi transfer Mobile-CRISPRi offers two...bacteria with a chromosomal copy of the RP4 transfer machinery mobilize these plasmids for transfer to recipient...contain a chromosomal copy of the RP4 transfer machinery, which mobilizes the Tn7 plasmids, and lack the...wild-type bacteria and the relative frequency of each of the 40 library strains was measured after 6 and...

...limited in the level of resolution that they can achieve. This means that regardless of how powerful of ..., you can’t resolve two points within ~250 nm of each other. For reference, individual presynaptic terminals...sample (i.e. cultured cells or tissues).  Step 2: attach anchors to your sample that will link biomolecules...evenly in all directions, aka isotropically. But achieving isotropic expansion depends on how well the gel...especially when paired with complementary experimental approaches. Since it arrived on the scene, ExM has helped...

...with many of the same drawbacks to each reagent type, so this approach is not suitable for all antibodies...and outlined the considerations to be made during each step. Now that you know the basic principles of ...the accessibility of your target epitope(s). To achieve these goals, researchers commonly use one of two...detergents, such as Triton X-100, Tween-20, or saponin. Each detergent has different chemical properties that...

...  Figure 1: (A) Components involved in each type of CRISPR-Transposon system. (B) Mechanism ...Recently, the lab introduced a streamlined expression approach (Fig. 2A), involving two-plasmid or single-plasmid... constructs. (B) High integration efficiencies achieved with VchINT, at two different incubation temperatures...insertions into multiple distinct targets could be achieved (Vo et al., 2020). VchINT was also shown to be...Zhou Z, Smock DC, Tang K, Owens TK, Krishnappa N, Sachdeva R, Deutschbauer AM, Banfield JF, Doudna JA (2020...

... to be able to cross the blood-brain barrier to reach the brain. Delivering the CREATE AAV library via...jumble of fluorescent neurons. A simple but flawed approach to multi-color labeling is to inject a mix of ...was derived from figure 3b in Chan et al.   To achieve the full diversity of colors AND sparse labeling...co-injected. The dose of this AAV can be adjusted to achieve sparse levels of labeling, without loss of color...virus, but the desired sparsity of labeling is achieved with lose doses of inducer virus. Additionally...

...-associated genetic dependencies associated with each ancestry group. Since about 90% of the cell lines...their data.     Fig. 1: Genomic fraction for each of five major ancestry groups for 994 of the Cancer...RNA that bind to a cell’s DNA, telling the Cas machinery where to cut.      Fig. 2: Heatmap indicating...): 250-252. Carlson J, Henn BM, Al-Hindi DR, Ramachandran S. Counter the weaponization of genetics research...  Rachel Leeson...

...spent this remaining time by elaborately pranking each other, dressing in costume, and holding competitions... we are sending hundreds of plasmid requests out each day. As you may imagine, it keeps all of us quite...furthering our mission by hard work, we genuinely enjoy each other’s ideas and company. The Addgene summer picnic...sometimes work is a bummer when you’d rather be at the beach or binging a new Netflix series. The thing that ...you can enjoy the people you spend 40+ hours with each week. In conclusion - get out there any have some...

...number of days and tracking average transit time for each courier we contracted with so we can always offer...can see our most popular educational resources for each type of resource we offer.  In 2013, we launched...experimental help. We love it when a researcher reaches out to ask if they can help us create or update... We’ve learned that increased accessibility is achieved through interconnected incremental changes. It... Rachel Leeson...

...Tracking cellular history with protein ticker tape Teaching an old antibody new tricks   New compact, efficient...REcipient Cells Through Envelope Design). This modular approach combines an interchangeable set of fusogenic and...side effects. This platform offers a promising approach for experimental therapies and medical applications... into the TurboID enzyme, Lee and Cheah et al. achieved precise control over when and where the enzyme... bioRxiv — doi: 10.1101/2021.10.13.463862.    Teaching an old antibody new tricks By Ashley Waldron While...

...by Kusumika (Kushi) Mukherjee, a Postdoc at Massachussetts General Hospital. A little over a decade back...non-integrating RNA virus Episomal vectors- extrachromosomally maintained DNA plasmids that express reprogramming...encoding reprogramming factors behave as separate extrachromosomal elements in the nucleus of the target cell...Epstein-Barr Nuclear Antigen-1 (oriP-EBNA-1) system where each vector contains a viral origin of DNA replication...Mukherjee is currently a postdoctoral fellow at Massachusetts General Hospital with research interests in ...

...This post was contributed by ATCC Outreach Scientist Nick Amiss. Cell culture in the present day Cell... number, cell line, and date, which you complete each time you passage the cells (as well as recording...liquid nitrogen for your Working and Master stocks of each cell line. Once all Master vials have been thawed...the results are reliable. With this two-pronged approach, the many mistakes of the past can be prevented...desired clinical endpoints.   Nick Amiss is an ATCC Outreach Scientist at LGC Group, London, which is the European...

...anneal with each other, while the 5’ flaps containing the original DNA sequence anneal with each other. Excision...identify dozens or hundreds of particles surrounding each of the highly-visible beads on the cryo-EM grid.... samples during cryo-EM grid plunge freezing By Rachel Leeson Cryo-EM samples can be damaged at the air-water...plunge freezing represents a convenient, effective approach to protecting your cryo-EM samples from damage...

...expression, and viability. Gates are the parameters the machine uses to differentiate between variations in those...tumor cells, immune cells, blood cells and more), each coming with a different size and structural complexity...A) A flow plot showing two stains in relation to each other. Using a quadrant gate, I can divide my cell...cell population into DN, DP and SP (for each stain respectively). (B) Instead of a quadrant gate, I am...occur when a cell is pulsed by the laser. Within each type of scattering, you also discovered that there...

... will overcome certain obstacles, alternative approaches, potential collaborations, critiques of your ...and all of the caveats and alternatives for an approach, if asked. The key to a chalk talk is to effectively... the knowledge gap in the field? How will your approach resolve this problem? (2-3 sentences) What.... Identify the key points you want to convey for each section or aim. Make sure those get priority. The... fluorescent proteins,  and live cell imaging approaches. Erik is also an avid long distance runner, gardener...

...obvious that a desired career outcome is not easily achieved. Of note, it was highlighted during the conference...Positive Points for Trainees - Taking Steps to Achieve a Successful Career Career Advice from the Career...showing researchers that they can take steps to achieve success in their careers. The take-away message...can take the time to do this, you should always reach out to your peers and other mentors who can help... postdocs, or professors isn’t nearly enough to reach diversity goals. Universities need to show that ...

...I have a list of what I really need in order to reach my research goals. Would I be able to get access...lack of resources, then ask for suggestions to meet each others’ needs and limitations. Everything is negotiable...research training to help you find other solutions to reach your goals. For example, a junior investigator turned...seem, you should consider it if it will help you achieve your research goals, even if that means saying ...a laboratory research management consultant and coach in Boston, MA. He specializes in assisting research...

...happening everyday, it’s not possible for us to reach everyone. Therefore, to make our library more useful...the repository? Let us know! You can notify us to reach out to potential depositors in two ways: 1. Fill...include any additional details that will help us reach out to the corresponding potential depositors. ...plasmid. While we cannot guarantee that everyone we reach out to will end up depositing, we hope this campaign...

...histone modification at specified loci. Charles Gersbach’s lab has deposited mammalian expression constructs...Both of these methods also recruit epigenetic machinery - but is there an advantage to using direct epigenetic...Vockley CM, Thakore PI, Crawford GE, Reddy TE, Gersbach CA (2015) Epigenome editing by a CRISPR-Cas9-based...Safi A, Song L, Hilton IB, Crawford GE, Reddy TE, Gersbach CA (2017) CRISPR–Cas9 epigenome editing enables...Ruan K, Wang F, Xu G, Hu R (2016) A CRISPR-based approach for targeted DNA demethylation. Cell Discovery...

...our bloggers each and every month, we've decided to start an "Editor's Choice" series. Each month, I'll...was a fantastic month for the Addgene blog. We achieved roughly 50,000 views over the entire blog and ... Expression in Mammals Recombinase-based State Machines Enable Order-dependent Logic in vivo As always...

... used in limited circumstances. An alternative approach is to flank the desired change with 4-5 silent...from mini-λ or 4) from the lambda red phage itself (each of these will be discussed in more detail below)...can be modified with any of the below described approaches.    Bacterial strain with integrated defective...requires drug selection.  One drawback to this approach is that it requires the generation of bacteriophage...modification into a different background, if needed. This approach is best suited for modifying plasmids or BACs ...