...system. Knowing that GFP’s  structure stabilizes a 4-hydroxybenzylidene imidazolinone (HBI)-like compound...same thing? Using the HBI derivative 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) with relatively...PMID: 26000751. PubMed Central PMCID: PMC4441765. 4. Guo, Peixuan AND Haque, Farzin. RNA Nanotechnology...target metabolite creates an aptamer that will only bind to DFHBI in the presence of that metabolite (Fig...approach less sensitive in most applications. Learn About Fluorescent Biosensors Light-up aptamers (or, fluorescing...fluorophore protein. Years later, Samie Jaffrey’s laboratory took advantage of their deep knowledge of the...Tips for Using FRET in Your Experiments Learn more about GFP Controlling Protein Activity with GFP Resources...

...of the 2A sequence and the next peptide downstream.4 The "cleavage" occurs between the Glycine and Proline...20(8):845-60. doi: 10.1089/hum.2008.188.) PubMed. 4. High cleavage efficiency of a 2A peptide derived ..., zebrafish and mice. Kim et al (PLoS One. 2011;6(4):e18556. doi: 10.1371/journal.pone.0018556. Epub 2011...genes above with your gene or genes of interest. Some of the plasmids listed in the table above (and their... an internal region of the mRNA. In the figure above, you can see that the IRES element acts as another... first discovered in picornaviruses, are short (about 20 amino acids) and produce equimolar levels of ...

... and Verkhusha (Nat Methods. 2013 Aug; 10(8): 751-4. doi:10.1038/nmeth.2521) Far-red light photoactivatable...fluorescent proteins. Krumholz et al (Sci Rep. 2014 Feb 3; 4:3939. doi: 10.1038/srep03939.)  ... absorbance of water, which absorbs wavelengths above 900 nm or so. In other words, wavelengths between...

...periods of time, especially at room temperature or at 4°C. Storing DNA: temperature and longevity You should...be stable for years. Plasmid DNA can be stable at 4°C or even room temperature for a short period, and...to purify DNA without a kit Read our blog posts about plasmid cloning Browse our Plasmids 101 blog posts...

... for more than making pretty pictures (see figure 4). It also enables single-cell anatomical study and...49 +/- 7% 36 +/- 8% 37 +/- 10% AAV.PHP.eB 69 +/- 4% 55 +/- 7% 76 +/- 5% Table 1: Transduction efficiency...induction of multi-color labeling of neurons (figure 4). These capsids can serve as powerful cell-type-restricted...or the PNS, respectively.  Read on to learn more about these two new AAV capsids and how they can be useful...Important considerations when using AAVs Learn more about using AAV as a tool for gene expression in mammals...

...had a tremendous impact on our world -- as of June 4, 2020, COVID-19 has caused an estimated total of 380,000...deaths worldwide (statistics provided by Google on 6/4/20). Many countries entered lockdown for weeks to ...during weekends and significant holidays. Figure 4: Plasmid request trajectories suggest an exponential...in his laboratory. Learn more here. Additional resources on the Addgene blog Learn more about plasmid...

...Membranes 3 mCherry 587nm/ 610 nm human β-Actin Actin 4 Citrine 516nm/ 529nm human α-Tubulin Tubulin Repeatable...methods. 2009. May;6(5):343–345. Pubmed PMID: 19363495. 4. Josse J, Kaiser AD, Kornberg A. Enzymatic synthesis...Approaches in Mammalian Systems. PLoS One. 2015 Apr 24;10(4):e0124958. PubMed PMID: 25909630. PubMed Central PMCID...Resources on the Addgene Blog Read about Gibson Assembly Learn About Other Plasmid Cloning Techniques Plasmids...

...POI’s) are fused to TAs and DBDs (see Figure 2a) (4). These fusions are then co-expressed in a yeast strain...bacterial LexA DBD and the lacZ reporter gene (3, 4), there are now many variations, including many that...repressor. Cell. 1985; 43:729-736. PubMed PMID: 3907859. 4. Fields, S., Song, O. A novel genetic system to detect... Plasmids Read our Blog Post About Yeast Vectors Read our Blog Post About Tagging Yeast Genes ...

...an attL-containing entry vector.   Figure 4: Method B to create an entry clone: TOPO cloning the...vectors at once in the same tube. You can clone up to 4 DNA fragments, in a specific order and orientation...Technology: Advantages and Drawbacks. Clon Transgen 4:138. doi:10.4172/2168-9849.1000138 2. Hartley JL. ... expression systems. Keep reading to learn more about the Gateway cloning method and its advantages. An... on the Addgene Blog Try Out FastCloning Learn About Methylation and Restriction Enzymes Use Colony PCR...

...function in the environment where it will localize [4]. Most investigators are surprised to learn that FPs...biology."Current Protocols in Cell Biology (2005): 21-4. Pubmed PMID: 18228466. Pubmed Central PMCID: PMC2875081...Pubmed PMID: 6526011. Pubmed Central PMCID: PMC557822. 4. Costantini, Lindsey M., and Erik Lee Snapp. "Fluorescent...moxFPs over standard FPs for the reasons listed above. In addition, because the moxFPs are highly monomeric...Medicine. Her doctoral research was conducted in the laboratory of Erik Lee Snapp and focused on optimizing fluorescent...

...alternative approach is to flank the desired change with 4-5 silent changes in the wobble codons, i.e. make changes... changes to the third base pair of the adjacent 4-5 codons that alter the nucleotide sequence but not ...defective prophage 2) from a plasmid, 3) from mini-λ or 4) from the lambda red phage itself (each of these will...2016. doi:10.1128/ecosalplus. PubMed PMID: 27223821. 4. Sharan SK, Thomason LC, Kuznetsov SG, Court DL. Recombineering...currently a research scientist in the Department of Laboratory Medicine at the University of Washington. She... Additional Resources on the Addgene Blog Read about Homology Directed Repair Use Knockout/Knockin Plasmids...

... SpyCas had similar levels of editing. See figure 4 in Harrington et al for more examples of GeoCas9’s...with reaction buffer for 8 hours at either 37 °C or 4 °C. Substrate was then added and cleavage product ..., see the table below and read on to learn more about GeoCas9.   SpCas9 GeoCas9 Size 1368 AA 1087...vivo. What aspect of GeoCas9 are you most excited about? Thermostability? Longevity in human plasma? Something... Additional Resources on the Addgene Blog Read about CRISPR Methods for Bacterial Genome Engineering   ...

...transposon; the plasmid is then degraded by the cell.4.) The transposase creates a double strand break in...stable transgenic cell lines. Biotechnol J. 2015 Feb 4. doi: 10.1002/biot.201400821. PubMed. 2. Mates L, ...of DNA that can move around in a genome. In a laboratory setting, transposons can be used to both introduce...

... A flexible chemical tag. Nature Methods, 2(4), Article 4. https://doi.org/10.1038/nmeth749 Strebinger...HaloTags and SNAP-tags. Cell Chemical Biology, 26(4), 584-592.e6. https://doi.org/10.1016/j.chembiol.2019.01.003...Proximity-Induced Reactivity. ACS Chemical Biology, 4(7), 547–556. https://doi.org/10.1021/cb900062k Gautier...but it’s real! In this post, we’re going to talk about the power and flexibility of self-labeling protein...ready-to-use without any cloning at all! As mentioned above, several variants of these tags have been developed...Illuminating Choices blog post for more discussion about choosing a dye or ligand to use for your experiment...

...and chat with me remotely via an audio chat (Fig. 4).  Learn: This works great for on-camera presentations...back to producing protocol videos?   Figure 4: The remote directing setup, complete with wireless...camera control, and extra hand sanitizer.    Cycle 4: Venturing into the Lab Design: The lab is the beating... Production Specialist, I’m constantly thinking about ways to improve our productions to deliver the best...four months after I started with Addgene, I was about to have a check-in with my boss Jennifer. I had ... would be a couple months. Jennifer and I spent about an hour that morning scrambling. Everything we had...someone to be comfortable on camera while thinking about things like lighting, camera angle, and shot composition...

... successfully tested the pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli...control over copy number. MUCICAT system contains 4 plasmids: pDonor, pTnsABC, pQCascade and pCutamp.  ... constructed plasmids (pDonor and pQCascade) with 4 rounds of transformation to complete the N-acetylglucosamine...complete editing process. The MUCICAT system While the above systems are great for experiments that require only...serve to interrupt genes (or gene clusters) in metabolic engineering. So far, we’ve used the MUCICAT system...when compared to the industrial strain. In our metabolic engineering case study of N-acetylglucosamine ...Multiple induction systems to choose from. Ready for metabolic engineering with improved cargo dedicated to gene...

...extension time, 3) increased concentration of MgCl2, 4) increased concentration of dNTP, and/or 5) the addition...glycosylase (UDG) and Exonuclease III (ExoIII) (step 4). UDG recognizes uracils that are in DNA and ExoIII...Methods 13.11 (2016): 928-30. PubMed PMID: 27723752.  4. Fowler, Douglas M., Jason J. Stephany, and Stanley... all desired codon changes (step 1 in the image above). A second universal primer is used to synthesize...first followed by the top strand. Both produced about the same number of positive clones. See Supplementary... of DNA degraded in the first digest (step 1 in above diagram). Remember that the BbvCI enzymes nick the...mutagenesis with the same protocol. What do you think about one pot saturation mutagenesis? How would you use...

...fluorescence intensity was classified into four groups (4; brighter than control, 3; same as control (Cetn1/...you can hold off on genotyping until the pups are 2-4 weeks old and use the ear tag method (creating different...differentiation 56.1 (2014): 122-129. PubMed PMID: 24372541. 4. Wang, Haoyi, et al. "One-step generation of mice ...the pX330 plasmids used in the validation assay (above) and it is just as effective (Mashiko et al., 2014...Engineering with CRISPR Validate Your Genome Edit Learn about Non Homologous End Joining and Homology Directed...

...storing for less than a day, lentivirus can be kept at 4°C. For long-term storage, viral preps should be divided...expression profiles.” Biotechniques 35.4 (2003). 812-4. Pubmed PMID: 14579747. 3. Drexler, H.G., Uphoff, ...Cytotechnology 39.2 (2002). 75-90. Pubmed PMID: 19003295. 4. Uphoff, C.C., Drexler, H.G. “Detection of mycoplasma...Lentiviruses are a powerful laboratory tool often employed to establish cell lines that stably express...Get Tips for Titering Your Lentivirus Learn All About Viral Vector Elements Learn How to Do Genome-Wide...

...manipulate prokaryotic cells? Especially, if you only have 4 hours and 35 students! After a course coordination... the format I decided on was to have two teams of 4 people face each other on a single chosen clinical...manipulation more hands on While teaching courses about gene therapy and genetic manipulation, I and other...with some other professors, I went home thinking about how to solve this problem. I didn’t want to face...therapeutic strategies work. Find Addgene Blog posts about other educational tools Playing BUILD YOUR PLASMID...winner? Ultimately, society should be! This is a collaborative game, as it should be in real scientific practice...practice transferable skills like team building, collaborative critical thinking, and communication among others...