...Recently I learned that Addgene’s pLKO.1 cloning protocol is viewed around 3,000 times each month. I ...Addgene website in December 2006, the pLKO.1-TRC cloning vector (deposited by David Root of the Broad Institute...this plasmid, and if it still being used. After learning of it's continued popularity, I decided this protocol...scientists, publishers, and small start-ups are turning to software apps to solve our need for quicker,...protocol listed under “Supplemental Document”: gRNA_Cloning Vector (deposited by George Church’s lab). Addgene...has also cataloged a variety of useful molecular cloning protocols, including making LB agar plates, restriction...), and now Addgene. I just began uploading our cloning protocols to the site and will keep you updated...

...about pooled library screening, read our recent blog post on Genome-wide Screening Using CRISPR/Cas9. If...lentiCRISPR v2 is key to its efficient use in screening experiments; large amounts of virus are required...a single empty vector and in pooled libraries containing thousands of pre-cloned gRNAs. DNA aliquots of... If you need help cloning a gRNA into lentiCRISPR v2, you can take a look at the protocol kindly provided...Ophir, et al. "Genome-scale CRISPR-Cas9 knockout screening in human cells." Science 343.6166 (2014): 84-87... vectors and genome-wide libraries for CRISPR screening." Nature methods 11.8 (2014): 783-784. PubMed ...

...extract, and 5 g/L NaCl) or on LB-agar plates containing glucose and L-arabinose at 37oC for at least ...induced with arabinose and colonies on arabinose-containing plates will be visibly white, and gas vesicles...expression heterogeneity required single cell screening to isolate a clonal cell line capable of producing... gene silencing required addition of chromatic-opening agents to boost expression to appreciable levels...Anabaena GVs in mammalian cells without the shell stiffening protein GvpC, which allowed the GVs to be flexible...vesicle expression and robustness without chromatin-opening agents. It ended up being very straightforward ...cells for a several generations to dilute the unintegrated plasmids (Fig. 2b). Next, perform a short (12...

...recordings and lesioning studies have been essential for this task. However, most lesioning methods destroy...destroy areas larger than intended and may unintentionally remove connecting pathways between other distant...function, with far fewer side effects compared to lesioning methods -  they may be the key to further unraveling... brain areas associated) compared to an animal running to escape a threat. Locomotion can thus be used...transgenic animals and viral vectors, scientists are combining genetic manipulations with the forced swim test... as sleep, food intake, mobility/coordination, learning, and memory. We’ll cover these in a  future blog... Vincent Jean Pascoli, and Christian Lüscher. "Refining deep brain stimulation to emulate optogenetic ...

... process and tested it by running the main Nextflow script file (containing our new process) on our local...From the beginning, one of Addgene’s goals was to ensure the scientific community obtained high-quality...pipeline. Benefits of Nextflow for bioinformatics pipelining In many use-cases, including ours at Addgene,...a container is sequestered from other programs running on a computer, so it safely operates in isolation... to the ease of jumping back and forth between running the pipeline locally and in the cloud. Creating...print statement at the end of the script or by opening the files. By working on one process at a time,...and decreased cost compared to purchasing and maintaining our own infrastructure. Our patchwork collection...

... revolutionary discovery, including CRISPR, is turning potential into impact. In molecular and cellular... contradicting their initial assumption. Eugene Koonin and Kira Makarova, longtime collaborators of Omar... the project. Using computational observations, Koonin and Makarova found that Cas7-11 appeared to have...toxicity compared to other RNA editing methods — streamlining experimental design and its potential for clinical...reported no detectable effect on cell viability – meaning there was no measurable cell toxicity – using Cas7...integration was observed in non-dividing cells, meaning that this system wasn’t dependent on the cell’s...sequence and integrate it into any DNA sequence containing the 38-50 bp sequence recognized by the serine...

...electrophoresis (PAGE) followed by silver staining or SYPRO Ruby staining and the molecular weight and relative...after negative staining. Empty vector particles can be identified after negative staining and appear darker...of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV subjected to ...Microscopy The ratio of empty to full (i.e., genome-containing) AAV particles within representative vector preparations... Trono lab. Cell culture medium (OptiPro SFM) containing lentivirus is first cleared by low-speed centrifugation...followed by centrifugation. Precipitated pellets containing viral particles are then resuspended in PBS, ...

...species, explaining why they recognize different guide RNAs. Deep in the REC lobe, the arginine-rich bridge... we can see why: two arginines make base-specific hydrogen bonds to the guanines, but the N (a thymine...guide RNA is bound primarily by the REC lobe. An arginine-rich bridge helix connects the two lobes and helps...stem loops bind along the outside of the protein, spanning both lobes. Stem loop 1, nestled between the REC... (yellow) make base-specific hydrogen bonds to guanines of the PAM in the non-target DNA strand (dark ..., 2022; Sternberg et al., 2015). The correct positioning of the non-target DNA strand is important for...DNA strand, and Cas9 bends the DNA helix by transitioning between the open and closed conformations. This...

...that have standard cloning sites or gateway compatible cloning sites for easy cloning of your genes of ...interneurons in mice as well as other organisms, opening the possibility of using these tools in virtually...ability to be coexcited along with EGFP while retaining an emission spectrum that is easily separable ...editing, Rees et al. created HF-BE3, a base editor containing high fidelity Cas9 variant HF-Cas9. They subsequently...reprogramming  Regulation of telomere length with TZAP Maintaining telomere length is important for cellular function...function and genomic stability, as telomere shortening leads to premature aging and cancer. The Denchi ... and repair templates of choice though modular cloning steps. The Voytas lab plans to update the toolkit...

... and find plasmids containing these fluorescent proteins at Addgene. 7. Designing Your Chalk Talk for... Hot Plasmids - March 2019 - Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable...Proteins by Cary Valley, Shreya Vedantam, Michelle Cronin, and Angela Abitua Thanks to a team effort from...101: Positive and Negative Selection for Plasmid Cloning by Jennifer Tsang There are many strategies for...systems and sacB counter-selection) tools used in cloning. 8. Bright Monomeric Fluorescent Proteins: mNeonGreen...chalk talk for an academic job interview including defining your research vision, organizing your vision into...

...at the Broad Institute, John Doench. A genetic screening project can be a tremendous undertaking, producing...sad. Learn More About CRISPR Pooled Library Screening Why the kinome? While properly designed perturbations...technology, gRNAs to knockout genes – are essential screening components, the model system and the assay are.... The individual plasmids will be useful when screening for phenotypes, such as cell-non-autonomous effects...effects, that do not lend themselves to screening in a pooled setting. The individual plasmids will also...imaging or gene expression assays necessitating the screening of individual plasmids separately. Additionally... will find these reagents to be useful. Happy screening! Many thanks to our guest blogger, John Doench...

...provided with a detailed training schedule that helped me transition easily. Training and responsibilities...responsibilities Through this training, I was gradually introduced to Addgene as a company and given more details on...on what my position entailed. The official training process was lined out to span the first three months...months of my six month employment. In the beginning, I spent a lot of time shadowing members of my team ... what I was to do for my time at Addgene. This training prepared me for the independent work on the QC...concern to the plasmid function. The steps of the training process gave me enough time to become familiar...

...will introduce you to a few useful labeling and staining techniques beyond standard surface labeling. Pro... Pro tip! Many people use 'labeling' and 'staining' interchangeably to refer to any technique that combines...Addgene uses 'labeling' to refer to antibodies and 'staining' to refer to small molecule dyes. Indirect labeling...labeling must be performed only after viability staining and surface labeling are already done. Intracellular... viability. Unlike intracellular labeling, dye staining can be done on live cells. For cellular proliferation...cell, the ester group is cleaved off by enzymes, turning the resulting molecule fluorescent and membrane-impermeant... for specific downstream applications, while maintaining a clean cell surface, dump gating is a useful...

...Agrobacterium-compatible, Cas9 containing vectors ready for Golden Gate mediated cloning of your gRNA of interest...researchers to insert targeting oligos with minimal cloning required. As with other model systems, multiple...promoters (Belhaj et al., 2013). Addgene carries Cas9-containing plasmids for knockout, activation and repression...on the helper plasmid and used to insert the intervening region into the plant genome. Created with BioRender.com...deaminase-mediated base editing (cytosine base editor or adenine base editor) and reverse transcriptase-mediated... et al., 2019). Both cytosine base editors and adenine base editors have been developed for plants and...Demirer GS, Zhang H, Ye T, Goh NS, Aditham AJ, Cunningham FJ, Fan C, Landry MP (2019) DNA nanostructures...

...alleviate those differences, they had a hard time maintaining their friendships. I put “neutral” in quotations...learn how to sell them. Types of Conflict Before explaining my approach to conflict resolution, it’s important... into a gap between teams that nobody is really owning. Conflict in this latter scenario usually manifests...disagreeable, you recognize their feelings as valid. Listening: In one of my favorite books, Michael Ende’s Momo... nearby village (and to the reader) by simply listening. Chances are, you know someone like this, even...conflicts.  Institutional Knowledge: Of the tools I’m mentioning here, this may be the most controversial, so ...also aggravate long-term personal conflicts.  Transitioning a conflict into a collaboration may not be the...

...commonly, DSBs are repaired by the nonhomologous end-joining (NHEJ) pathway, leading to nonspecific nucleotide...Furthermore (3), we have developed a framework that, by combining these two strategies allows you to specifically...find the 0.2-0.5% correctly edited alleles, when screening reasonable amounts of single cell clones. Furthermore...Furthermore, CRISPR/Cas9 editing is often bi-allelic, meaning that even if one allele is accurately edited, the... few hundred. From these two steps, you’ll be screening a total of a few hundred clones - Still better...longer feasible to incorporate mutations without screening thousands of clones to find a positive one. So... single cell. We calculated the likelihood of obtaining homo- and heterozygous mutations by multiplying...

...that there was no cross-reactivity, opening the door for immunostaining analysis. To achieve even cell labeling..., but can be detected via immunostaining. This feature facilitates screening of Cre-negative Brainbow ...systems. Pre-Brainbow techniques, such as Golgi staining, injection of diffusible labels, or electron microscopy...segment, eliminating two color possibilities. The remaining cassette can invert as long as Cre remains expressed...Second, the system did not permit analysis by immunostaining. Although the fluorophores used are fluorescently... Cai et al. also created an Autobow construct containing both Cre and XFPs. Cre production drives recombination...

...demonstrates that transitioning from academia doesn’t have to mean abandoning it or running away screaming...weren’t some pretty hefty bumps during my graduate training. I particularly think that too many young scientists...talking to people about science and that I love explaining things to people. When I started my graduate ...keeps me on top of a variety of fields thereby broadening my knowledge. In addition, this position allows...careers outside of academia during my graduate training. PhDs have many roles to play inside, on the outskirts...

...their goal of PhD training when selecting a research topic. The end point of training is not to become ...After joining a lab, the focus shifts to choosing an unexplored and impactful research topic that aligns...consider when choosing a research project After joining a lab, the next important decision to make is “...what approach will I take and what can I learn, learning the difference between good and bad data, and ... dreams. He also highlights the importance of combining “new discoveries or technologies increase chances...Advice for Choosing a Research Project Finding and Joining Your Dream Lab Advice for Choosing a Research Project...

...scientist-in-training will spend 10 or more years with a small number of formal advisors learning how to be...priorities will help in determining a good fit. Here are just a few suggestions for determining whether an advisor... will be ones that help them succeed after the training period. While there are many aspects to choosing...time hanging out with members of the lab. It is a warning sign if the PI does not welcome you to visit and...Precision and Plasticity in Mentoring" Don’t ignore warning signs If lab members, neighbors or alumni give ...