...more foolproof (still without a kit!) As mentioned above, working with RNA requires keeping your samples ...lysis. This can be challenging depending on your laboratory situation or tissue collection method, so biotechnology...supernatant is extracted with chloroform as in the above no-kit method.  Researchers have also developed ...

...Narrowing the field After working your way through the above steps, you may find that you still have a number...advice if you need to troubleshoot. What do you know about the vendor and the vendor’s technical support? An...validation is equal and you should think critically about what data is and is not shown on an antibody website...

...students as we have done in the past. Below, read about the five winners and their research.   Abhi Aggarwal...work has only been made possible due to the collaborative and highly interactive environment that Janelia...biosensors or biosensors that detect new redox metabolites are vital for researchers to better understand...

...  Read about another common type of antibody for research, monoclonal antibodies Learn about the advantages...Once the initial antibody response starts to wane about a month later, the animal is given additional booster...

...make AAV ideal for human therapies and in vivo laboratory experiments ranging from transient gene expression...genetic cargo. Typically, when researchers speak about AAVs, they are actually referring to rAAVs. ITRs...Primer for Beginners Read Our Lentivirus FAQ Learn about Viral Vector Elements Resources on Addgene.org ...

...Check out our post on Biotinylation to learn more about biotin and its use in molecular biology. Other Conjugates...perfectly blissful, productive lives without thinking about these chemical reactions. After all, there is an...ready to use off the shelf. So why are we talking about this? Even though there are so many commercially...

...which can be used with this approach - read more about them on our CRISPRa blog! Fig. 1 CRISPR ... is a lot of overlap with the systems discussed above, just with more elements of control. Tet promoters...to be site-specifically knocked in. This can be labor intensive and time consuming. If your native protein...

... is smaller than the traditional SpCas9 enzyme (about a third of the size) and does not require a tracrRNA...Cas12a to further optimize it for therapeutic and laboratory usage. Fast facts – PAM requirement: TTN or ... the lack of collateral cleavage). You can read about Cas7-11 in our blog post on its development and ...

...“I hope it isn’t degraded” when scientists talk about their precious RNA samples. RNA is inherently less...efficacy as well as the unnecessary stress of worrying about the status of your RNA. gRNAs modifications are ...Some of the RNA stabilizing modifications listed above were developed before CRISPR-Cas9, for use in applications...

...is useful for many metabolic engineering and synthetic biology goals. In metabolic engineering, the expression...or can interfere with the precision needed in metabolic engineering or other synthetic biology circuits...

...With this new found structural knowledge, in collaboration with the Joung Lab, they used targeted mutagenesis...edits in mammalian cells and may decrease worries about off target effects in applied and/or therapeutic...at Addgene. Resources on the Addgene Blog Read about Cas9 Homolog Cpf1 Read How Non-Homologous End Joining...

...process.” The ClonTracer library originally came about as Bhang and co-workers were trying to develop a...particularly interested in applying what we have learned about tumor heterogeneity to drug discovery and the development...Additional Resources on the Addgene Blog Learn about the Sabatini and Wood Drug Screening Kit Find Out...

...interpretation of the results? Then think deeper about the data they are showing. Are those experiments...points of the protocols. Are there any red flags about how the work was performed that could influence ...University. She is a fan of photosynthesis and writes about autotrophs and biochemistry on her blog. Follow ...

...streptomycin, tetracycline, combinations of the above, or other. High or Low Copy Choose from: high...the “Purpose” field. If you ever have a question about what should go into a cell, hover over the cell ...Addgene and contact your technology transfer office about the MTA. Pro Tip! Download a new spreadsheet each...

...homozygous alleles at both of your genes of interest. About 1 in 4 mice will be homozygous for the mutation ...Find our Plasmids 101 blog posts Read blog posts about using Cre-lox Read our genome engineering blog posts... posts Resources on Addgene.org Find more about Cre-lox Read our Genome Engineering Guide Learn more...

...of their choice in mammalian cells.  Learn more about Mammalian shRNA Tools for RNAi! Cre-Lox technology...knockdown. Vector provided by the Qi lab. Learn more about CRISPR plasmids for transcriptional inhibition. .... Check our CRISPR guide and blog to learn more about CRISPR and the most recent developments in the genome...

...glycosylation (CDG) are a group of rare inherited metabolic diseases with severe and life-threatening consequences...understood.  In line with this, Prof. Videira and collaborators will use Addgene plasmids to immortalize CDG...discovery led them to investigate the use of a common laboratory chemical, guanidine hydrochloride, as a potential...

...Vectors Featured Topic Page Learn about CRISPR-AAV vectors Learn about Addgene AAV Quality Control Process...viral infections. She is particularly passionate about HIV cure research and science communication.     ...

...more blog posts about base editing Learn about another evolved base editor: ABE8e Read about xCas9, a CRISPR...

...Addgene blog Read more about using AAV for retinal gene therapy Find more about precise neuronal targeting...injected into macaques and postmortem human retinas. About 25% of these AAV synthetic promoters successfully...

...Daniel M. Sapozhnikov, a graduate student in the laboratory of Dr. Moshe Szyf at McGill University. Some ...expression, confounding any potential conclusions about the role of methylation (Hrit et al. 2018, Kaas ...Daniel M. Sapozhnikov is a graduate student in the laboratory of Dr. Moshe Szyf at McGill University, working...

...posts Read blog posts about neuroscience Resources on addgene.org Learn more about chemogenetics in our...as a DREADD agonist due to its ability to be metabolized into clozapine (CLZ). While some scientists argue...

...specifically refers to the organizational schema described above, and not any particular cloning technique. Though...imagine, there is an array of distinct goals within metabolic engineering, synthetic product production, and...MoClo strategy and kit if you want to learn more about this cloning technique! Many thanks to Rob Hurt ...

... primer binding site, starting with a length of about 13 nt (Anzalone et al., 2019). Primer binding sites...of reverse transcriptase template, starting with about 10–16 nucleotides. For longer templates, it is even.... Test multiple nick sites, starting with sites about 50 bp upstream and downstream from the prime editing...

...inducible expression systems that allow tuning of metabolic pathways. Fertility (F) plasmids: microbial sharing...available in the Addgene repository. To learn more about some other common plasmid features, such as origins...Auriol, P., Vogel, T. M., & Simonet, P. (2001). Laboratory-scale evidence for lightning-mediated gene transfer...

...allowed gene circuits to be deployed out of the laboratory in a cell-free and biosafe format that was stable...circuit-based sensors to be used outside of the laboratory. The detection threshold for these RNA sequences...sequences (3 nM), however, was quite high and above what would be expected in patient samples (e.g.: blood...

...grown at 30 °C instead of 37 °C. If you’re unsure about the integrity of your virus, perform a diagnostic...Additional Resources on the Addgene Blog Learn All about Adenovirus Associated Virus New to Virus? Check ...Check out Our Primer for Beginners! Learn All about Lentiviral Vectors Additional Resources on the Addgene.org...

... Blog  Read about Other Means of Genome Engineering Using FLEx AAV Vectors? Learn about Viral Vector ...12665802. 2. Branda, CS and Dymecki, SM. Talking about a Revolution: The Impact of Site-Specific Recombinases...

...inserted GFP is expressed (knocked in). As seen in the above example, you can remove a floxed resistance gene...Get the Basics of Homology Directed Repair Read about Generating Knockouts with CRISPR through Non-Homologous...Expression Vectors Find Cre-Lox Plasmids Learn More about CRISPR  ...

...a workspace with plasmid, not that I would know about this… A no template control (NTC) should always ... variability. Check out this post to learn more about different ways to titer AAV. Do you have any AAV...at Fred Hutchinson Cancer Center for teaching me about AAV production and purification! References 1. Aurnhammer...

...tagged gene. Most insertions can only be detected by labor-intensive processes like PCR or by evaluating visual...repair after a CRISPR/Cas9 induced break. Learn more about these constructs and the cell lines they’ve been...Additional Resources on the Addgene Blog Learn about Fluorescent Protein Timers Check out Photoactivatable...

...with current 3C methods provide useful information about chromatin interactions, they are grainy so it’s ...interactions opens the door for answering more questions about chromatin conformations. The Xu lab presented three...Cas9 Variant for Your Next Experiment Learn More About Designing gRNAs Against Your Favorite CREs Here ...

...Do you ever wonder about the origins of some of the common techniques or tools you use in the lab? Take...times of the myc tag.  Origins of protein tags For about as long as the disciplines have existed, cell and...examples of new versions of Myc1-9E10 mentioned above basically involve taking the Myc1-9E10 variable ...

...articles; so if you are interested in learning more about any one of them, please click on the links throughout...laying around the lab. If not, you’ll be looking at about $50-$60 per enzyme. Once you’ve got a few of these... relatively inexpensive and can be obtained for about $70, resulting in a price of a few dollars per reaction...

...viral genome sequencing (VGS). Why should you care about viral quality control? AAV vectors are produced ... let us know if you have any specific questions about viral DNA NGS you’d like us to discuss. We are also...or at [email protected] to answer your questions about our quality control process! The images in this...

...sold by Clontech and is in a freezer box in just about every lab in the world, is not inert. In all seriousness....11 mM [2]. Following the simple logic outlined above, you might think EGFP is unlikely to form dimers...affinity assays used in the papers describing the FPs above including molecular sizing columns and native gels...

...preparation from Addgene, you can skip the steps above! Cells are then transduced with lentivirus containing...practices, CRISPR libraries can help you learn a lot about your phenotype of interest. For more detailed information...Libraries” (McDade et al., 2016). To learn a bit about how CRISPR/Cas9 can be used in other types of experiments...

...such as full plasmid sequences provided by the laboratories that deposit their plasmids with us or other...mismatches and gaps in the alignment. If you are curious about the differences in the blastn programs, check out...Inside Look at NGS Plasmid Quality Control Learn about our Snapgene-powered plasmid maps. Resources on...

... J. (2002) Arabidopsis : a laboratory manual. Cold Spring Harbor Laboratory Press. Available at: https...https://books.google.at/books/about/Arabidopsis.html?id=IfZAMNPWVk4C&redir_esc=y (Accessed: 21 September ...

...CRISPR Featured Topic Page Learn about RNA Targeting with Cas13a Learn about CRISPR DNA Base Editing Resources...and sequences that can be edited.   As discussed above, the ability to edit RNA has multiple advantages...

... droplet generator, you will have to be careful about transferring your droplets from the droplet generator...Additional resources on the Addgene blog Read more about polymerase chain reaction Browse our blog posts ...collection of viral vector related protocols Learn more about the Addgene viral service ...

...Blugene (check back on December 5 to learn more about this!). I’ve assembled a series of screenshots ...search. These pages provide helpful information about the subject, link to related guides and protocols...Additional resources on the Addgene blog Learn more about other features on Addgene's website ...

...by replicate values provides useful information about the accuracy of the curve, which we will discuss...many scientific applications users aim for an R2 above 0.95.    Additional considerations When running ... or make smaller dilutions.        As mentioned above, it is a good idea to run the standard dilution ...

...organizations are working to help educate the public about proper antibiotic usage. Although these steps are...laudable, they likely won’t solve the growing problem. Above all, we need new antimicrobials, ideally ones for...used to deliver any of the three systems discussed above, with the caveat that phages are very diverse, and...

...Korea, and beyond!   Check Out How Addgene Thinks About Developing Lab Management Software    Organizing...of other scientists using pLKO.1 variants learn about those updates? This is a problem many scientists...our Plasmids 101 blog series. Want to learn more about sharing your plasmids via Addgene? Check out all...

...Plasmids 101 Learn about genome-wide screening with CRISPR and Lentivirus Read about the ClonTracr Library... the proper cell line is used; several common laboratory cell lines including 293 were immortalized with...

...Generating a transgenic plant takes even longer, is more labor intensive, inefficient, and often not reproducible... region is transferred into the plant. In the laboratory we can utilize this natural process by replacing...system. The vectors mentioned in this blog post above are ready to use vectors, but with the MoClo system...

... more about CcdB, the toxic key to efficient cloning Read all Plasmids 101 blog posts Read about knockout...with the other toxin-antitoxin systems described above, one part of the pair is more stable than the other...

...following unanswered questions about CRISPR screen design. Unanswered questions about genome-wide CRISPR/Cas9...Morgens et al drew some generalizable conclusions about off-target activity, demonstrate safe-targeting ...

...successfully to derive iPSCs from PBMCs. Most laboratories use Sendai virus to reprogram PBMCs efficiently...establishment of virus-free hiPSCs usually require about 5-10 passages. Furthermore, working with Sendai ...source of somatic cells. This translates to only about 16 hiPSC colonies per 100 ml of starting blood sample...