...studies have shown that the conditions Sotos-type 2 and Marshall Smith Syndrome are due to mutations in...accurate diagnosis of NFIX-related syndromes (Sotos-type 2 and Marshall-Smith) and could be expanded to NFIA-...Rescue occurs through guanidinium cation (Gdm+) binding in the location formerly occupied by the Arg sidechain...

...excise themselves from one location and then insert somewhere else (Fig. 2). Class II TEs have LTRs on...on both of their ends.    Figure 2: Overview of DNA transposon transposition. Modified from Sandoval-Villegas...has four major components: 1) a CRISPR RNA (crRNA), 2) four proteins (TniQ, Cas8, Cas7, Cas6) that forms... It generally stays put, which makes it easy to locate in a genome. But there is a type of mobile DNA,... hyperactive and likes to jump around from one location in the genome to another. This jumping is what...most transposable elements (TEs) results in the duplication of this target site sequence at the site of insertion...This is similar to how retroviruses, like HIV, replicate. Class I TEs do not encode a transposase enzyme...

..., and the desired gene (transfer vector plasmid), 2) the HIV-1 genome containing all viral genes with ...protocols that have been described (protocol 1, protocol 2, protocol 3) to find the one that will give you the...PubMed PMID: 3785217. PubMed Central PMCID: PMC367857. 2. Naldini, Luigi, et al. "In vivo gene delivery and...mammalian cells often relied on chemical (CaCl2, cationic liposome) or electrical (electric current forming...particles and the virus particles produced could not replicate. However, Mother Nature found a way to re-create...packaging plasmid forming a vector that could create replication competent virus. Even though this system was ...still a potential risk for the generation of replication competent lentiviruses (RCL) especially if you...

...handing them your application 3 days before the deadline isn’t going to be much use. 2. Originality is ... it’s important not to throw in the towel if 1 or 2 funding bodies say no. If possible, get feedback from...Series 4. Become a science communicator More and more fellowship applications are asking for a 200-300 ...good quality application. Even following this advice, only about 50% of my grant applications have been ...do need to be able to research and write your application, then give it to someone else to read and / or...it). It’s easy for reviewers to spot when an application is rushed (whether the idea isn’t properly researched...researched / developed or the application is full of typos) and these are unlikely to get past the initial...

... reduce the background staining (Table 2).      Table 2: Secondary antibody selections for a four-antibody...your experiment (all well validated for your application, of course!) When the stars are aligned and the...factors that affect isotype choice. Target and application should be considered The first question you may... a strong signal and to work in a variety of applications. The desired specificity of your secondary antibody...affect my experiment?” Well, it depends on the application and target. If you are running a basic western...target is expressed at very low levels or your application requires a high degree of affinity, then species...considered when choosing an antibody. In some applications probing species-on-species, i.e. using a mouse...

...and the Journal of Emerging Investigators. Chapter 2 - Getting high school students and teachers interested...Additional Resources from the Addgene Blog Find education resources Use our Plasmids 101 eBook to teach ...

...FRET signal back again. The Ting lab constructed 2 versions of this reporter to target the K9 (pcDNA3... 525.7570 (2015): 533-537. PubMed PMID: 26352475. 2. Bygren, Lars O., et al. "Change in paternal grandmothers...Mechanisms of epigenetic modifications The main mechanism of epigenetic modification is chromatin remodeling...literally means ‘on top of the genome’). Epigenetic modifications can result in phenotypic changes without any...any change in genotype. Epigenetic modifications play a large role in turning genes ‘off’ and ‘on’, signaling...two main mechanisms, either through histone modifications, which can influence the transcription of genes...hypomethylation, on the other hand, has also been implicated in the development and progression of cancer ...

...Hrit et al. 2018, Kaas et al. 2013). Figure 2: CRISPR TET-based epigenetic editing. In reality, ...approach for targeted DNA demethylation. Cell Discov 2, 16009. https://doi.org/10.1038/celldisc.2016.9.  ...approach (spoiler alert: it’s dCas9… with no modifications). Ready to get started?   Figure 1:... when DNMT1 methylates the genome during DNA replication, it doesn’t methylate sites blocked by dCas9....for dCas9 to interfere with DNMT1 even through replication. The dCas9 also needs to be removeable, otherwise...vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat Biotechnol 34, 1060–1065. ...

...from another within a fully stained sample. Figure 2 is an example of the emission spectra for two fluorophores...another during spectral unmixing.     Figure 2: The full emission spectra for BV421 and BV711 on ... tumors, and moreso than other methods, the identification and isolation of rare cell types. In recent...complexity index ranges from 0-1, with a value of “0” indicating those markers are completely distinct from one...one another, and a value of “1” indicating those markers are identical and completely indistinguishable...

...Joachim ended up at the University of Amsterdam Chapter 2 - Joachim’s Academic and career goals. Career advice...Twitter and has helped us update many of our educational resources. Listen to hear all about Professor...speeds science. Chapter 8 - The importance of communicating research findings through social media and other...

...from animals or hybridomas.   Figure 2: (Left) Figure 2: (Left) The light chain and heavy chain genes...cells, for antibody production and subsequent purification.  Academic laboratories and companies have already...Edition. 2104. Cold Spring Harbor Laboratory Publications, New York. Helma J, Cardoso MC, Muyldermans ...

...due to the presence of additional helper sequences (2-3). As alluded to above, termination and polyadenylation...and translation processes as well.   Figure 2: The conserved eukaryotic polyadenylation signal directs...type are generally broken down into two broad categories, prokaryotic or eukaryotic, based on the functional...termination mechanisms fall under two general categories: rho-dependent and rho-independent. Rho factor...

...important role in solving the structure of DNA. 2. The Immortal Rap of Henrietta Lacks Students in Oakland...advice on how to dive into new forms of science communication. Click here to listen to the interview with ... from Addgene.      Check out more science education tools like Verse Two and Fill in the Planck at...with us. Tom McFadden is a YouTuber and science educator looking for creative ways to implement the next...next generation science standards and communicate science. Learn more about Tom on his youtube channel ...Community Check out All of Our Podcasts Find Great Educational Content in our Plasmids 101 Series Additional...

... Zetsche et al., Nat Biotechnol. 2015 Feb 2;33(2):139-42. doi: 10.1038/nbt.3149. Light-inducible ...and on your favourite signalling pathways (Figure 2). They are all available now at Addgene. 1 Lungu...20;19(4):507-17. 10.1016/j.chembiol.2012.02.006. 2 Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; ...Nihongaki et al., Chemistry and Biology. 2015 Feb 19; 22(2):169-74. High efficiency cloning for enzyme screening...inducible, gene knockdown with shRNA or shRNA-miR30, (2) tamoxifen-inducible CreERT2 gene deletion, (3) simultaneous...different fluorescent proteins, 8 constitutive promoters, 2 includible promoters, 3 polyA terminators and various...of plasmids required for each input/output system; 2) removing all chemically inducible promoters; 3) improving...

...scientists have 1) the courage to make mistakes and 2) the integrity to recognize these mistakes quickly...She studies beta-cell function and failure in Type 2 Diabetes. Birmingham, AL, USA 33.5207°N 86.8025°W ...available to people who want to join the dialog and 2) remember that science should not be partisan: everyone...also a science communicator, UBC Public Scholar, and a member of the Vancouver SciCATs. Follow her on ...researchers and supporters of science marched to advocate for scientist participation in government, evidence...promote help motivate you to spend a little time advocating for science and getting yourself out there for...April because devaluing science has alarming implications for our public policies—many of which impact...

...Accessible Scientific Writing" on Sunday, from 12 pm - 2 pm. If you want to learn about, and practice, principles...Finally, our team is rounded out with our science communication specialist (you'll know me by my Tweeting — ...

...26678082. Find the plasmids from this publication at Addgene. 2. Dalvai, Mathieu, et al. (2015). “A Scalable... PITCh vector via In-Fusion or SLIC cloning. Step 2: Design an insertion locus-specific gRNA The gRNA ...another method for making targeted modifications. A recent publication by Zhang et al. shows just that -...about a month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction...required by MMEJ can easily be added via PCR amplification. Given the inefficiency of HDR for knock-ins... and 3’ junctions, respectively. These results indicate that PITCh is a robust method for genomic insertion...lies in the diversity of nucleases and their applications. It will be interesting to see what new editing...

...NGG-3′ (“N” can be any nucleotide base, followed by 2 guanines). This limits the use of ABEs as DNA sites... in the promoter region of gamma hemoglobin (HGB1/2) leads to continuous expression of fetal hemoglobin... from E.coli, named TadA7.10 in ABE7.10)  a catalytically impaired Cas protein (dCas or Cas nickase)  ...accidental insertions and deletions (indel) by the catalytically impaired D10A nickase from ABE7.10 can be a ...the authors created ABE8 constructs using a catalytically “dead” S. pyogenes Cas9 (dC9-ABE8.x-m/d). The...these variants, ABE8 could target many additional locations in the genome. Compared to ABE7.10, NG-ABE8.xm... reduced off-target editing frequencies. For applications that require high DNA editing specificity, for...

...from GFP and the mutation definitions found in Table 2 may not apply. Table 1: The specific mutations comprising..., V163A EBFP F64L, S65T, Y66H, Y145F   Table 2: Functional role of specific mutations in GFP derivatives...transgenic mice. Purification: GFP can be used as a general epitope tag for protein purification and a number...their experimental needs, and some of the common applications in the lab. Why Green Fluorescent Protein?...to maintain fluorescent activity; very little truncation is tolerated, however, point mutations are acceptable...Re-engineering GFP to increase its range of color and application Almost as soon as its sequence was elucidated...others) Neutral mutations    A multitude of applications Due to its size and ease of use, GFP and other...

... digest and compare it to some reliable controls. 2. Viral stocks may be sensitive to freeze thaw cycles...PMID: 24003179. PubMed Central PMCID: PMC3760253. 2. Rohwer, Forest. "Global phage diversity." Cell 113.2...experiment and they don't do anything. Viruses are complicated and they perform complex biological tasks, both...vector may undergo genomic rearrangements during replication. To avoid genomic rearrangements, try amplifying...membranes (Davis et al., 2002). Polybrene is one such cationic reagent that is commonly used and has been shown... during transfection, and does not necessarily indicate the production of virus. Alternatively, if the...hepatitis B virus DNA and hepatitis C virus RNA quantification as measured with branched-DNA technology." ...