...iSRIRACCHA) where Cas9 expression is activated using 4-OH-T and doxycycline. A gRNA that targets your desired...found that H2B-GFP expressing cells exhibited a 2-4 fold increase in the number of endogenous indel edits...off target effects. However, these methods can be labor intensive and quite time consuming.   As Cas9/gRNA...have been several systems to do this but most are labor-intensive, require multiple fluorescent proteins... Browse all of our CRISPR blog posts Find more about fluorescent proteins Get a refresher on CRISPR with...

...Retroviruses and cancer. Rev Infect Dis. 1982 Sep-Oct;4(5):1015-25. DOI: 10.1093/clinids/4.5.1015 Lee C.S....Era of Personalized Medicine. Genes Dis. 2017 Jun;4(2):43-63. DOI: 10.1016/j.gendis.2017.04.001 Maetzig...clinical settings. AAV is also a cool tool in the laboratory! It is ideal for whole animal administration,... be helpful to you. Interested in learning more about a specific virus? We have you covered with guides..., adenovirus, and AAV. If you’re wondering more about what applications these viruses are good for, we...

... multiple plasmids (3 for 2nd-generation systems, 4 for 3rd-generation systems). The components of both...Safer. Replication incompetent and always SIN: Uses 4 plasmids instead of 3 and eliminates the requirement... outlined in the simple schematic to the right. 3-4 plasmids are transfected into A293T cells: one transfer...Cronin J, Zhang XY, Reiser J. Curr Gene Ther. 2005. 5(4): 387-398. PubMed . HIV-1 Genome Nuclear Import Is...Donello JE, Trono D, and Hope TJ. J Virol. 1999. 73(4):2886-92. PubMed . A Third Generation Lentivirus Vector...Production Schematic Frequently Asked Questions (FAQ) about Lentiviral Plasmids What is the difference between...lentiviruses, please review 2nd vs 3rd generation above . Briefly, 2nd generation lentiviral systems use...

...adaptive immune responses, which flies do not have!) (4). The power of Drosophila as a model organism is in...PMID: 22038952. PubMed Central PMCID: PMC3677821. 4. J. R. Huh et al. Digoxin and its derivatives suppress...immunofluorescent images like in this paper (2). Metabolic assays such as Seahorse can be employed to look...Additional Resources on the Addgene Blog Learn about Using Cre in Drosophila Read Other Genome Engineering...

... mechanism, but their small packaging capacity of 4-5 kb limits the delivery of larger transgenes, such... toxicity in long-term experiments.  Figure 4: The C-terminus of Cre (CreC) and the N-terminus of... plasmids you need. If you'd ever like to write about a recent plasmid deposit please sign up here.    ...tracing using the transgenic zebrafish model, ubi:zebrabow. In this system, cells express various fluorescent...

...and 70186, respectively). Antares Chu, J., et al. (4), likewise, wanted to produce a BRET probe for imaging...modular base-specific gene tagging using a ligase-4-dependent mechanism. Nature Comm. 7, 12338. PubMed...PMID: 26424696. PubMed Central PMCID: PMC4668208. 4. Chu, J., et al. (2016) A bright cyan-excitable orange...Blog Learn more about FRET Tips for Using FRET in Your Experiments Learn more about Fluorescent Protein...heme-containing proteins in whole animals, light at or above 600 nm wavelengths should be used. Researchers often... emits light at a tissue-penetrating wavelength above 600 nm. LumiFluors Schaub, F.X., et al. (3) designed...be excited by NanoLuc® luciferase (NLuc) and set about to create an in vivo imaging reporter by linking...

...pAAV-CA-FLEX Cre activates gene of interest, using 4 mutated lox sites AAV Uchida Cre Reporters and Tools...Recombination Between loxP Sites. J Mol Biol. Aug 25;1504(4):467-86. doi:10.1016/0022-2836(81)90375-2. PubMed ...Learn about the Cre-Lox system and how it can be used for genetic rearrangements and recombination. Use...

...tubes, and would prevent the pH change upon thaw (Fig.4). To test this, we froze the tubes at -80°C and then...transport or storage on dry ice. Nat Methods. 2013 Apr;10(4):278-9. PubMed PMID: 23538862. 2. Potter M, Lins B...return the sample back to the original pH. What about putting it back in the freezer? We then tested whether...

...under a variety of conditions. They are composed of 4 proteins encoded in a single operon and were first... Find Plasmids from This Publication at Addgene 4. Petroni, G., Spring, S., Schleifer, K.-H., Verni,... Additional Resources on the Addgene Blog Read About Other Hot Plasmid Technologies Read Other Synthetic...

...Cell Stem Cell 4, no. 6 (2009): 472-76. PubMed PMID:19481515. PubMed Central PMC: 2705327  4. Hockemeyer,...reprogramming à la carte." Nature Reviews Genetics 12, no. 4 (2011): 231-42. PubMed PMID: 21339765. Methods figures...PubMed PMID: 20139967. PubMed Central PMCID: 2892897. 4. Stadtfeld, M., and K. Hochedlinger. "Induced pluripotency...methods do you have experience using? Let us know about your iPSCs experiences in the comments below! References...Additional Resources on the Addgene Blog Learn about Methods to Reprogram Peripheral Blood Cells Check...

...expectations. (Don't think you can finish that book in 4 weeks!)” “Sometimes a mentor is a sounding board--...Connie Cepko to learn about her mentoring style. Top 7 questions scientists ask me about mentoring In this... answers to the top questions scientists ask me about mentoring. Here they are: What makes a good Mentor... Where can I find a Mentor? What is this I hear about “group” or “peer” mentoring? How do I ask someone...a good science mentor? I started a conversation about many of these topics with the researchers who joined...often one that devotes time and energy to thinking about how to be a great Mentor.    Check Out Angela Depace's...questions in future posts. If you have a question about mentoring that you want us to cover, let us know...

...small multiplex experiments 4-color flow panel: BV421, FITC, PE, and APC 4-color microscopy panel: DAPI...protects mucosal membranes Dimer with a valency of 4 IgA1; IgA2 IgD Coexpressed with IgM on the surface... As a guideline, most antibodies can be stored at 4 °C for short-term storage. For long-term storage they...conjugated to fluorophores should never be stored below 4 °C. Certain isotype subclasses, such as IgG3, aggregate...aggregate after thawing and therefore should be kept at 4 °C. Record and track lot numbers for all antibodies...typically use HRP as a signaling molecule. Figure 4: A direct sandwich ELISA, which uses a capture antibody...are often used in clinical applications. Diabodies - Diabodies contain two Fab fragments, each recognizing...

...futures both inside and outside of academic research (4). There are currently 17 sites around the USA undertaking...PMID: 22567149. Pubmed Central PMCID: PMC3342243. 4. Mathur, Ambika, et al. "Transforming training to ... the Jackson Laboratory for Genomic Medicine and a Senior Research Associate at the Labor and Worklife...increasing data collection about the scientific workforce, in particular about postdocs. Among other involvements...Research Symposium Boston 2015 – Academic Data and the Labor Market. For more information on the symposium as...generating too many trainees per PI” “Lack of awareness about how the system operates and functions” “Complete...available in academia (5, 6). Academic data and the labor market for PhDs Later this week, postdocs and students...

...carbohydrates added to antibodies post-translationally. 4) Modifications made to antibodies, such as inclusion...Heterogeneous Populations to Defined Reagents. Antibodies 4:197–224. https://doi.org/10.3390/antib4030197  Hegazy...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...

...Alternatively, glassware can be baked at 180°C for at least 4 hours.   If your final RNA sample(s) are resuspended...time it took to isolate RNA from 20+ hours to around 4 hours, and variations on this no-kit method are still...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 ...

...DNA, then they could diminish off-target cutting (4). However, instead of weakening interaction sbetween...PMID: 25079318. PubMed Central PMCID: PMC4176945. 4. Kleinstiver, Benjamin P., et al. "High-fidelity CRISPR–Cas9...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...

...homozygous alleles at both of your genes of interest. About 1 in 4 mice will be homozygous for the mutation at ...mice, as well as progeny from future generations. 4. Continue breeding your colony You’ll want to generate...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...

...Encouraging students to do science anywhere.  Chapter 4 - JEI's mission and how they assess whether or not...working in a lab for the first time, and thinking about the future. Many thanks to our guests,Olivia Ho-Shing...resources Use our Plasmids 101 eBook to teach students about basic plasmid biology Use or CRISPR 101 eBook to...

...killer gene”. Gene. 1994 Oct 11;148(1):71-4. PubMed PMID: 7926841. 4. Bernard, P., et al. “The F plasmid CcdB...in modern cloning, and why should you learn more about it? Read on to find out how ccdB can make your cloning...of this system for enhancing cloning efficiency about 20 years ago and developed cloning vectors to harness...

...Find the plasmids from this publication at Addgene. 4. Kiani, Samira, et al. “Cas9 gRNA engineering for ... and repression.” Nat Methods. 12(11) (2015):1051-4. PubMed PMID: 26344044. PubMed Central PMCID: PMC4666719...Additional Resources on the Addgene Blog Read More about Fluorescent Protein-Based Tools Learn Some of the...