...phosphoserine directly into recombinant proteins (5). Optimization of this system defined three components...into recombinant proteins, such as the kinase MEK1 (5). MEK1 is inactive without requisite dual phosphorylation...307.5717 (2005): 1969-1972. PubMed PMID: 15790858. 5. Park, Hee-Sung, et al. "Expanding the genetic code... one of the MEK1 phosphomimetic residues in the above study with phosphoserine (to create a pSer/phosphomimetic...non-phospho species on PhosTag gels as described above. Reagents and protocols Sep-OTSλ plasmid - Addgene...interested in the intersection of cell signaling, metabolism, and the mighty mitochondrion. You can follow...Blog Verify Your Constructs with Colony PCR Read about Tags for Protein Purification Resources on Addgene.org...

...downstream of the promoter, you can add an EcoRI site just 5’ of the start of the gene and a HindIII site just ...two HindIII digested ends will anneal leaving the 5’ end of your gene just downstream of the promoter ...plasmid : insert ratio” in the ligation reaction. 5. Purify the finished plasmid   Once it looks like... (post or pre run in the table above, respectively). Some of the above stains require you to visualize...equivalent to backbone + insert, unsuccessful 2 and 3 above), you can try ligating the recipient plasmid alone...get experimenting!   Additional Resources: Read about Gel Stains on the BitesizeBio Blog  Additional ...

...makes it so forgiving, is the incorporation of a 3’-5’ exonuclease in the reaction mixture. The exonuclease...expression cassettes for yeast transformation can take 5 days. NEBuilder shortens that time to one day by simplifying...scarless plasmid inserts.  Want to Learn More about DNA Assembly? Check Out Our Plasmids 101 eBook! ...to produce sgRNA for CRISPR experiments can be laborious, requiring multiple cloning steps. You can simplify...Additional Resources on the Addgene Blog Learn More about Gateway Cloning Gibson Assembly and Other Long-Homology...

...restriction enzymes that cut DNA at specific sites (5). Imagine that you have a genome with unknown sequence... allowed for many seminal scientific discoveries (5, 8). While newer techniques have largely replaced ...PMID: 19283728. PubMed Central PMCID: PMC2743315. 5. Roberts, Richard J. "How restriction enzymes became...abundant in a sample. Whereas Southern blotting is labor intensive and requires a large amount of high-quality...DNA technologies that are now commonplace in laboratories. As scientific technologies rapidly progress...PMC4411503. Resources on the Addgene Blog Learn More about Restriction Cloning Get Tips on Analyzing DNA Sequencing...

...recent paper to see the Gal4/UAS system in action (5). I use the Gal4/UAS system to knock down gene expression...Drosophila, 3, 1–10 (2015). PubMed PMID: 21356876. 5. J. Chow, Z. Marka, I. Bartos, S. Marka, J. C. Kagan... use the Gal4/UAS system, and you can read more about them in review articles (2–4). You can also take...

...recognizes the PAM sequence 5’-NGG-3’, where N can be any base. In Figure 5, we can see why: two arginines...complementary strand is mismatched.   Figure 5: S. pyogenes Cas9 bound to guide RNA and target DNA... Complex with Guide RNA and Target DNA. Cell, 156(5), 935–949. https://doi.org/10.1016/j.cell.2014.02.001...nascent heteroduplex and abolish cleavage (Pattanayak et al., 2013). As mentioned above, however, Cas9 sometimes..., 2022), indicating that there is more to learn about this process!      Figure 3: Crystal structure...rest of Cas9 and is highly flexible; it can rotate about 140 degrees between inactive and active conformations...conformational changes that make Cas9 such a valuable laboratory tool are still an area of active research. New...

...Zhang et al. designed multiple gRNAs to target the 5’ long terminal repeat (LTR) of the HIV-1 genome, which...Kaminski et al. designed gRNAs to target the HIV-1 5’ and 3’ LTRs and expressed them along with Cas9 in...Replication and Accelerate Viral Escape. Cell Rep. 2016 Apr 5. PubMed PMID: 27068471. Ramanan V, Shlomai A, Cox ...reservoirs, chronic inflammation, and negative metabolic effects of ART. Why can’t the body mount an immune...

...provides both the endonuclease and ligase function while 5’ adenosine overhangs left by Taq perfectly anneal ...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...

...Advice on Picking a Graduate Lab and Project Check Out 5 Science Videos from Tom McFadden Resources at Addgene.org...of our Hot Plasmids podcast series, you'll learn about new red fluorescent proteins, AAV tools for targeting...

...Advice on Picking a Graduate Lab and Project Check Out 5 Science Videos from Tom McFadden Resources at Addgene.org...of our Hot Plasmids podcast series, you'll learn about optogenetics tools for controlling protein activity...

...Addgene Blog 5 factors to help you choose the right cloning method Find blog posts about scientific sharing...scientists save even more!) If you assume an average of 5 hours of hands-on time per plasmid, Addgene, since...cloning before you can start your experiment? What about all the reagents you need to order? Sometimes, it...dive deep into generating the data you’re excited about. When we were closing in on the one million plasmids...million plasmids shared than not!), we think it’s about time we updated this post. How much time does ...

...LTRs define the limit of what will be packaged. The 5’ LTR and 3’ LTR, respectively, act as RNA polymerase...adenovirus types, but most vectors are based on adenovirus 5 (Ad5).     Fig. 3 - Viral vector components...aren’t reflected in the common components outlined above for gamma-retroviruses. While not required for lentiviral...and helper plasmids for all the viruses discussed above (gamma-retrovirus, lentivirus, adenovirus, and adeno-associated.... Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997. Principles of Retroviral Vector ...

... Antibody Validation proposed 5 pillars for antibody validation.  The 5 pillars include genetic strategies...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...

...set at .05, then 5% of the time the statistical test will be incorrect. If you have 5 samples and perform...comparison is needed, without a high number of samples (~5). Tukey’s test – Most conservative test. Higher false...a summary snapshot without telling you anything about outliers or data spread within those data sets. ...the rules” and which we have certain information about. The assumptions and requirements that need to be...Quantitative If your data doesn’t fit the requirements above, a non-parametric test may be the way to go, which...only performing the comparisons you really care about. Family-wise error is the probability of a false...rate than the multiple comparison tests described above. Violated assumptions and statistics Statistical...

...transcription systems in Drosophila.” Nat Neurosci 9(5) (2006): 703-9. PubMed PMID: 16582903. Potter, Christopher...Caenorhabditis elegans.” Nat Methods 9(4) (2012): 391-5. PubMed PMID: 22406855. PubMed Central PMCID: PMC3846601...promoters.” Proc Natl Acad Sci U S A. 80(1) (1983): 21-5. PubMed PMID: 6337371. PubMed Central PMCID: PMC393301...can be used in the repressible manner described above, as well as the inducible manner detailed in our...necessary to have all 3 of the different binary systems above if they all behave very similarly?” The simple answer... at the same time.  The 3 binary systems listed above are orthogonal, meaning that they do not affect ...

...part A of the transgene contains a promoter and the 5’-fragment of the transgene, followed by a splice site...part A of the transgene contains the promoter, the 5’ half of the transgene, a splice donor sequence and... degron, or degradation sequence, upstream of the 5’ splice donor and downstream of the 3’ splice acceptor...first time AAV user Learn about how AAV is used in retinal gene therapy Learn about the parts of an AAV transfer...

...sequence and has been suggested to cause cytotoxicity [5]. Second, instead of using a donor-plasmid, you could...PMID: 23723351. PubMed Central PMCID: PMC3690869. 5. D. Grégoire and M. Kmita, “Recombination between ...retroviral insertion sites.,” Nat. Protoc., vol. 4, no. 5, pp. 789–98, Jan. 2009. PubMed PMID: 19528954. PubMed...transposase itself, please read on to learn more about this exciting technology from the folks at Transposagen...Additional Resources on the Addgene Blog Learn about the Sleeping Beauty Transposase Read up on Genome...

...modifications, specifically ones which do not contain a 5’ triphosphate in the gRNA, reduce the innate immune...Controlled CRSIPR-Cas-based Genomic Regulation. ACS 6, 5, 695-703 (2020). https://pubs.acs.org/doi/10.1021/...“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...

...assess whether or not their achieving it. Chapter 5 - JEI's future - getting more involved in the classroom...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...

.... 1993 Dec 5;234(3):534-41. PubMed PMID: 8254658. 5. Bernard, P. and Couturier, M. “Cell killing by the...ccd operon in plasmid F.” J Bacteriol. 1989 May;171(5):2353-60. PubMed PMID: 2651399. PubMed Central PMCID...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...