...scientific Twitter list were excluded from the dataset. 3. 60% of scientists on Twitter are male and 40% are...with scientific domains <10% of the time. Figure 3: Top 20 Domains Tweeted by Scientists. 6. The top ...use Twitter professionally to network, learn more about alternative careers for scientists, and share cool....” This paper addresses the following questions about scientists on Twitter: who are they? What do they...

...2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010 167  Nature 3.3%  10.5%  3.2  534.1 3 2010 ...plasmids in our data were published in Nature, but only 3 percent of papers without Addgene plasmids were. Thus...journal-adjusted citation measure we re-weight the 3 percent of Nature papers without Addgene plasmids ...2010 3 Applied and Environmental Microbiology  1.6%  1.2% 0.75   2.3 4 2010 167  Applied and Environmental...deposited at Addgene still accrue on average 2 to 3 times as many citations as other papers in the same... with plasmids deposited at Addgene – typically about four times as many as papers without plasmids deposited...

...preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in both distribution and GC-content, which might.... Additionally, whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered...Cpf1 activity, except when they were present in the 3’ end of the target sequence (bases 19-23). Cpf1 is...gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA target sequence does not have an ...decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target sequence. Cpf1’s strength may lie... Find plasmids from this publication at Addgene. 3. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker...System. Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 10.1016/j.cell.2015.09.038. PubMed PMID: 26422227...

...engineering has come. For comparison, in 2007 it took about 3 months to knockout a single allele in a human line...thing ask incredulously, “You seriously didn’t add 3 microliters of 5 mM star anise?” This is something...expression was across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal...stable cell pool were then expanded for an additional 3 weeks under blasticidin selection. Anti-Cas9 Western... control (WT). Vinculin is a loading control. 3. In terms of making the monoclonal line: I had never...with many additional tips can be found here. Figure 3: A surprise for the author. Checking Cas9 expression...would still be able to identify the cleavage product. 3. The Surveyor kit had a lot of good tips for first...

...preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in both genomic location and GC-content. After...for a surprise! Whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered... AT-rich regions or AT-rich genomes that lack the 3’-NGG PAM sites favored by SpCas9. Since Cpf1 doesn...Cpf1 activity, except when they were present in the 3’ end of the target sequence (bases 19-23). Cpf1 is...gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA target sequence does not have an ...decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target sequence. Figure 2: Effects... Find plasmids from this publication at Addgene. 3. Kim, Daesik, et al. Genome-wide analysis reveals ...

...Optogenetics Boyden 83898 pAAV-mDlx-ChR2-mCherry-Fishell-3 mDIx Activator Optogenetics Fishell 65014 pAAV-hsyn-Jaws-KGC-GFP-ER2...serotype from the Alla Karpova and David Schaffer laboratories. These viral vector preparations were produced...Janelia Research Campus viral project team in collaboration with David Schaffer (University of California...

...presence of additional helper sequences (2-3). As alluded to above, termination and polyadenylation of Polymerase...transcribed, and typically occur directly after any 3’ regulatory elements, such as the polyadenylation ...is non-specific and can be added to any accessible 3' end. The presence of the poly(A) tail targets the...releases the mRNA from the polymerase and creates a free 3' end which is now available for polyadenylation. The...Landick R. Bacterial Transcription Terminators: The RNA 3′-End Chronicles. Journal of molecular biology. 2011...are required for efficient rabbit beta-globin mRNA 3' end formation. Cell. 1987. PMID: 3568131.  Hager ...how the Promoter Controls Gene Expression Learn about New Cloning Techniques Read all of our Plasmids ...

...TRICK system, the 3’ UTR of the reporter mRNA is labeled by PCP-mCherry (Figure 2). The 3’ UTR also contains...protein (NLS-PCP-GFP) an RFP protein that binds to the 3’ UTR of the reporter mRNA via an MS2 coat protein ...multiple hairpin sequences into the coding sequence and 3’ UTR. Before beginning experiments, it’s important...PMC4451088 Find plasmids from this paper at Addgene. 3. Yan, Xiaowei, et al. “Dynamics of Translation of ...Additional Resources the Addgene Blog Learn More about SunTag Catch up on Your GFP Background Choose the...

... DNA by joining 5 ́-phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably...the ligation junction (the base pair providing the 3 ́-OH terminus to the ligation) than on the downstream...reviews 106.2 (2006): 687-699. PubMed PMID: 16464020.  3. Ellenberger, Tom, and Alan E. Tomkinson. "Eukaryotic...Colette, Veronique Bailly, and Walter G. Verly. "Nicks 3′ or 5′ to AP sites or to mispaired bases, and one-... And what is ligase fidelity anyway? Let’s talk about it. DNA ligases are enzymes that seal breaks in ...to accelerate characterization of nucleic acid metabolic enzymes." Nucleic acids research 44.2 (2015): ...

...relationship between deletion size and frequency [3]. There are several applications in which the production...to deletions following paired DSBs is inversions [3]. These rearranged segments may also be informative...is likely to bear an indel or inversion mutation [3]. In the CRISPR era, the genomic landscape may be ...level. Science 2013 Oct 11;342(6155):253-257. PubMed. 3. Canver MC, Bauer DE, Dass A, Yien YY, Chung J, Masuda...) [1]. Our initial work was with TALENs, in collaboration with the Porteus lab [2]. With the advent of...PhD student at Harvard Medical School in the laboratories of Stuart Orkin and Daniel Bauer. He received...

...identified 53 potential candidate genes that fell into 3 categories based on the architecture of the CRISPR...C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, but they require...with 5' overhangs Cas13a (C2c2) 2x HEPN crRNA RNA 3' A, U, or C (not required by all orthologs) Specific...analogous to the PAM sequence for Cas9, is located at the 3’ end of the spacer sequence and consists of a single...27669025. Find plasmids from this paper at Addgene.  3. Gootenberg, Jonathan S., et al. "Nucleic acid detection...Orthogonal Type VI-A CRISPR-Cas Enzymes. " Mol Cell. 66(3) (2017):373-83. PubMed PMID: 28475872.  Find plasmids...potential applications of Cas13a given what we know about its structure and function? For starters, Cas13a...

... or period of work): What 3 things would you say worked out best? What 3 things worked least well? ...different): Here are the 3 things I would say worked out best.  Here are the 3 things that worked least...employee about what they do well and where they need to improve, and they’re also clear about how the ...enough to tell them about areas they need to improve in. If a manager has concerns about an employee and ...language to talk about real performance issues. Watch this little video to learn more about indirect language...Medical School researcher Connie Cepko to learn about her management style. Formalizing the feedback session...informal feedback is a very effective way to bring about change in behaviors and habits in a nonthreatening...

...Monitoring Autophagy from Yeast to Human. Autophagy,3(3), 181-206. doi:10.4161/auto.3678. PubMed PMID:17224625...PMID: 26215030. PubMed Central PMCID: PMC4517063. 3. Mizushima, N., Yoshimori, T., & Levine, B. (2010)...Methods in Mammalian Autophagy Research. Cell,140(3), 313-326. doi:10.1016/j.cell.2010.01.028. PubMed ...17224625. 5. Noda, T., & Klionsky, D. J. (2008). Chapter 3 The Quantitative Pho8Δ60 Assay of Nonspecific Autophagy...information about the contents of the autophagosome nor does it provide information about what is being...higher pH of the vacuole. Read on to learn more about prior methods for studying autophagy and how Rosella... (as opposed to giving more fine-grained detail about what proteins or organelles are being degraded) ...

...1, 9 Svoboda 83898 pAAV-mDlx-ChR2-mCherry-Fishell-3 Dlx ChR2 mCherry Constitutive 1, 9, rg* Fishell 135634...and Optogenetics Guide page for more information about this field. Use the table and filters below to find...

...indicating that there is more to learn about this process!      Figure 3: Crystal structure of S. pyogenes...so crystal structures like those in Figures 2 and 3 don’t show these regions, even if they are present...other into the “closed” conformation shown in Figure 3. Though Cas9 is not a static molecule in any binding... RNA in this structure does not include stem loop 3, and guide RNA nucleotides 11-20 are not resolved ...required for Cas9 nuclease function. Stem loops 2 and 3 are not strictly necessary for in vitro cleavage, ...-20 are disordered and don’t appear in the Figure 3 crystal structure, reflecting the less stringent recognition...mismatches in this region. The crystal structure in Figure 3 shows the guide RNA stabilizing the DNA-binding protein...

...are closer to hiPSCs than age-matched fibroblasts [3]. These advantages make PBMCs the ideal source of...yielded hiPSCs at a low efficiency (0.001-0.03%) [3, 9]. In 2011, Chou et al. generated a mixture of two...PMID: 27579041. PubMed Central PMCID: PMC4989082. 3. Chou, B.K., et al., Efficient human iPS cell derivation...and gene expression signatures. Cell Res, 2011. 21(3): p. 518-29. PubMed PMID: 21243013. PubMed Central... and peripheral blood cells. Stem Cells, 2013. 31(3): p. 458-66. PubMed PMID: 23193063. 8. Ehrhardt, A...vectors for gene therapy. Curr Gene Ther, 2008. 8(3): p. 147-61. PubMed PMID: 18537590. 9. Mack, A.A.,...successfully to derive iPSCs from PBMCs. Most laboratories use Sendai virus to reprogram PBMCs efficiently...

... Genet. 2009 Jun;41(6):753-61. Epub 2009 May 3. PubMed. 3. Singh H, et al. A new approach to gene therapy...an endonuclease reaction occurs which cuts the DNA.3.) The released transposon can now bind a strand of... overhangs depending upon whether they are on the 3’ or 5’ end of the gene of interest allowing for insertion...of DNA that can move around in a genome. In a laboratory setting, transposons can be used to both introduce...

...the researchers did not have access to a P3 laboratory (BSL-3), a high-security facility needed to perform...suitability and availability; 3) Make the contact to start a direct collaboration between researcher and expert...our blog post about remote collaborations Read our Transferable Skills Guide article about external collaborations...needed. In the example above, for example, we fostered a formal scientific collaboration between two research...project. We think that these collaborations add enormous value. A discussion about a protocol can save weeks...approach was useful in humans. Then, one of them heard about our online platform, called Crowdfight, and filed...just the right infrastructure and offered to collaborate with them. The partnership was fruitful and a...

...the supernatant of lysed bacteria as template; and 3) run your PCR product on a gel to analyze product ...step to colony PCR is designing primers. There are 3 strategies for primer design: 1) insert-specific primers... primers, 2) backbone-specific primers, and 3) orientation-specific primers.       Insert-specific ...and swirl in a small amount of sterile water.  Pick 3-10 colonies in total to test, depending on the number...negative clones. Finally, orientation-specific primers (3) give the same band (+) or no band (-) result as insert-specific...SLIC cloning Learn about Gibson Assembly Additional Resources on Addgene.org Read about Choosing a Molecular...this type of primer does not provide information about the orientation of the insert. Orientation-specific...

...that information in the report as well.    Figure 3: An antibody data report, showing the results of an...methods section for the data report shown in Figure 3.   At the end of the report, you’ll find a results...Figure 5: Results for the data report shown in Figure 3. The antibody is rated "Pass". Curating the data ... antibody worked under the conditions described above, and a short description of how the antibody performed...

...): Where in the tissue/which cell type? Location (3): Traveling from the original location - Axonal Transport...regions where other serotypes may perform better (3). Brain Region  Optimal Serotype Striatum AAV5...(5), AAV6 (6) and AAV5 at excitatory connections (3). These facts beg the question: is axonal transport...Once thawed, the AAV aliquot should be used quickly. 3. Patience Is Bitter, but Its Fruit Is Sweet Time If... be determined what gene expression looks like at 3 days vs 7 days vs 14 days etc. To decrease the time...type of research, as they are safe to use in vivo. 3) The use of AAVs in humans AAVs are already being ...Therapy 16.6 (2008): 1073-1080. PubMed PMID: 18414476. 3. Aschauer, Dominik F., Sebastian Kreuz, and Simon ...

...desired gRNA target sequence, as well as the 5’ and 3’ homology arm repair template (Fig. 1, Step 1). There...marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type will produce... proteins in a tissue specific manner, as well as 3-site destination vectors for inserting a tag at multiple...genes in human cells. To use CRISPaint, users need 3 vectors:  1) a gRNA vector that targets the gene of...to specify the reading frame of the insertion, and 3) a vector containing the desired tag, which can be...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...

...lentiviral vector plasmid in Escherichia coli Stbl3. 3 Biotech 3:61–70. https://doi.org/10.1007/s13205-012-0070...be >12 kb. However if it recombines at the 5’ and 3’ LTR regions, it splits into two smaller plasmids.... Figure 2: An uncut plasmid is expected to form 3 bands: nicked, linear, and supercoiled. Here we find...molecular biology and viral vector protocols Learn about the common lab E. coli strains Find tips for viral...

...observed in the absence of genome editing (Fig. 2 and 3) (5, 3). Figure 2: Gene activation with nuclease-competent...its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation...alterations observed at the target site.   Figure 3: Gene repression with nuclease-competent Cas9. When...methods 12.9 (2015): 823-826. PubMed PMID: 26167643. 3. Kiani, Samira, et al. "Cas9 gRNA engineering for ...of the Addgene Scientific Advisory Board. His laboratory at Columbia University is focused advancing scientific... Listen to Our Interview with Alex Chavez Learn about Sequencing Options for CRISPR Genotyping Cas9 Activators...

...equivalent to backbone + insert, unsuccessful 2 and 3 above), you can try ligating the recipient plasmid alone... of the start of the gene and a HindIII site just 3’ of the end of the gene. This way you can then cut...DNA as this will be useful for the ligation step. 3. Ligate Your Insert into Your Vector In the ligation...recipient plasmid : insert ratio” of approximately 1:3. Since the number of base pairs for each varies, it...equivalent numbers of colonies on each plate (unsuccessful 3).     If you have a high number of colonies on your...colonies and check them for successful ligation. Pick 3-10 colonies depending on the number of background ... (post or pre run in the table above, respectively). Some of the above stains require you to visualize...

...bands at ~3.6 kb and ~1.1 kb (lane 3). Uncut DNA (lane 2) has a band at <3 kb, which corresponds to the supercoiled.... Mol Biotechnol. 2008 Nov;40(3):252-60. doi: 10.1007/s12033-008-9082-3. Epub 2008 Jul 8. PMID: 18607781...plasmid sample. Troubleshooting Tips   Figure 3. Summary of plasmid multimerization troubleshooting...9(4 PT 2):707-16. doi: 10.1016/0092-8674(76)90134-3. PMID: 797459. Berg CM, Liu L, Coon M, Strausbaugh...Behavior of Plasmid DNA. Microbiol Spectr. 2015 Apr;3(2):10.1128/microbiolspec.PLAS-0036-2014. doi: 10.1128...colonies using one of the detection methods described above can help ensure the isolation of the monomeric version...plasmid DNA production platform incorporating low metabolic burden seed-stock and fed-batch fermentation processes...

...premises we can classify a plasmid preparation in 3 different ways: Transformation grade DNA Cloning ...be extracted from small bacterial cultures (i.e. 2-3 ml) with or without commercially available kits and... backbone. Transfection grade DNA Figure 3: Need to introduce plasmids into mammalian cells? ... cloning Read our Plasmids 101 blog posts Learn about the differences between transformation, transduction...

...primer sequences! Primers consist of two regions: the 3’ end binds template DNA for PCR amplification, while...vector sequence between modification sites (see Figure 3), producing multiple fragments with specific homologous...introduced in a gene, while adding a GFP-tag (Figure 3). Achieving multiple modifications requires multiple...not easy to amplify by PCR, so we usually add DMSO (3%) and Betaine (1 M) to the PCR mix. When PCR is not...PubMed PMID: 8346047. PubMed Central PMCID: PMC331480. 3. García-Nafría, Javier, Jake F. Watson, and Ingo H... Watson and Javier García-Nafría from the MRC Laboratory of Molecular Biology. Plasmid cloning is an essential...with successful recombination reported in all laboratory E. coli strains tested so far. The use of this...

... 2] or even form obligate tetramers (i.e. DsRed) [3]. This is a problem for fusion proteins. One of the...potential to form large stable polymers of dimers. 3) FRET biosensors that contain two dimerizing FPs can...localize incorrectly and may function improperly. 3. Incorporation into a FRET biosensor A FRET biosensor...DsRed or high affinity FPs such as TagRFP. Problem 3 can be solved, in most cases, by using monomeric FPs...2002. 296(5569): p. 913-6. PubMed PMID: 11988576. 3. Matz, M.V., et al., Fluorescent proteins from nonbioluminescent...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...

...manipulate, 2) they have long-term expression; and 3) they have limited toxicity. However, a key challenge...1x1011 vs 1x1012 viral genomes (vg) per mouse. Figure 3 shows a gross comparison of AAV.PHP.eB and AAV.PHP.S...transduction, measured via GFP expression, was determined 3 weeks later. Table was derived from figure 2e in Chan...transduction, measured via GFP expression, was determined 3 weeks later. Table was derived from figure 3b in Chan...PubMed PMID: 26829320. PubMed Central PMCID: 5088052. 3. Lentz TB, Gray SJ, Samulski RJ. Viral Vectors for...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...

...why is it necessary to have all 3 of the different binary systems above if they all behave very similarly...multiple genes at the same time.  The 3 binary systems listed above are orthogonal, meaning that they do... synthetic systems. Recent work has even used all 3 in a single modular vector for ease of use! Conclusion...313. doi: 10.1007/s00253-018-9485-4. Epub 2018 Nov 3. PMID: 30392122. Pfeiffer, Barret D., et al. “Refinement..., lineage tracing, and mosaic analysis.” Cell 141(3) (2010): 536-48. PubMed PMID: 20434990. PubMed Central...reagents for genetic manipulations.” Nat Methods 12(3) (2015): 219-222. PubMed PMID: 25581800. PubMed Central...can be used in the repressible manner described above, as well as the inducible manner detailed in our...

...length. It contains short promoter sequences at its 3’ and 5’ ends that are crucial cis-acting signals for...shortened version of the viral genome. It contains the 3’ and 5’ ends of the genome which are required for ...nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola virus...the viral polymerase initiates RNA synthesis at the 3’ end of the minigenome to produce the positive sense...Berlin Heidelberg, 2004. 1-41. PubMed PMID:15298166. 3. Hoenen, Thomas, et al. "Minigenomes, transcription...Microbiology, National Emerging Infectious Disease Laboratories.  She is interested in genome replication and...

...." F1000Research 3 (2014). Pubmed PMID: 25653845. Pubmed Central PMCID: PMC4304227. 3. Sauermann, Henry...positions within 5-7 years of earning their PhDs (3), showing that the ‘standard’ pathway into academia..., Eve. "Looking out for future scientists." eLife 3 (2014): e04901. Pubmed PMID: 25291257. Pubmed Central... 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...

...plasmids ready for deep mutational scanning.     3 reasons to be interested in deep mutational scanning... Taq polymerase, 2) increased PCR extension time, 3) increased concentration of MgCl2, 4) increased concentration...synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled template strand is degraded ...mutagenesis method are 1) ease of use, 2) time savings, and 3) ability to perform single site or multi-site saturation...Mutagenesis Protocols, pg. 103-109. PubMed PMID: 20676978. 3. Wrenbeck, Emily E., Justin R. Klesmith, James A. ... 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...

...the termini of the crRNA molecule (usually 1-3 of the first 3 bps are modified) for optimal stability and...crRNA also contains a ~20 bp repeat region on its 3’ end. This repeat region associates with the trans-activating...sugar ring, a popular stabilizing modification is 3’ phosphorothioate linkage, where a sulfur replaces...“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...

...antigen. J Gen Virol., 73 (Pt 3), 653–660. https://doi.org/10.1099/0022-1317-73-3-653. PMID 1372038. Randall...comparable to GFP during early embryogenesis (Figure 3). mScarlet-I3 also offered clearer signals due to ...autofluorescent tissues like the intestine. Figure 3: Expression of fluorophore-tagged GOLG-4 through eight...disruption in Mycobacterium abscessus. J Bacteriol., 206(3), e0033523. https://doi.org/10.1128/jb.00335-23.  ...more feasible, the lab of Alessio Ciulli and collaborators developed CRBNmidi (Kroupova et al., 2024). ...stepwise, achieving mutation efficiencies of up to about 9% of colonies (102 to 104 times higher than traditional...Bentley Time to get flexible! Alex Dunn’s lab and collaborators have deposited a new mechanosensor called PILATeS...

... step involves digesting phage genomic DNA into 1-3 kb (approximately gene-sized) fragments so that these...bulk of resultant fragments falling between 1 and 3 kb results in optimal likelihood of capturing a whole...filter paper and saturate each with lysis solution (~3 ml each), pouring off any excess. When your protein...Expression Vectors in the FX Cloning System Kit  Step 3: Probe random phage DNA fragments with live host cells...PMID: 27536293. PubMed Central PMCID: PMC4971087. 3. Simpson, David J., Jessica C. Sacher, and Christine..." Chemical Reviews (2016). PubMed PMID: 27319741. 3. Simpson, David J., Jessica C. Sacher, and Christine...our planet, but we still know collectively little about how they infect and influence the evolution of their...

...crRNACpf1 Endonuclease size spCas9: ~4 kbsaCas9: ~3 kb ~3.9kb crRNA/gRNA length gRNA: ~100 nt crNA:... Lastly, an AAV vector was used to multiplex edit 3-4 genes in a primary culture of mouse neurons and ...and GFP.  Of these neurons, ~15% had indels at all 3 targeted loci.  See the chart below for a summary ...Endonuclease of a Class 2 CRISPR-Cas System. Cell,163(3), 759-771. PubMed PMID: 26422227. PubMed Central PMCID...targets compared to Cas9.  Read on to learn more about Cpf1 multiplexing.  For an in-depth review of Cpf1... Addgene. If you have any questions or thoughts about Cpf1 multiplexing, leave them in the comments below...

...Recombinant DNA by CcdB.” Biotechniques. 1996 Aug;21(2):320-3. PubMed PMID: 8862819. 2. Bahassi, EM., et al. “F ...Microbiol. 1995 Mar;15(6):1031-7. PubMed PMID: 7623659. 3. Bernard, P., et al. “Positive-selection vectors using...DNA cleavage by gyrase.” J Mol Biol. 1993 Dec 5;234(3):534-41. PubMed PMID: 8254658. 5. Bernard, P. and ...DNA-topoisomerase II complexes.” J Mol Biol. 1992 Aug 5;226(3):735-45. PubMed PMID: 1324324. 6. Tam, JE. and Kline...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...

...Scientific reports 3 (2013). PubMed PMID: 24284873. PubMed Central PMCID: PMC3842082. 3. Mashiko, Daisuke...pCAG-EGxxFP target plasmid contains overlapping 5′ and 3′ EGFP fragments under the ubiquitous CAG promoter....classified into four groups (4; brighter than control, 3; same as control (Cetn1/sgRNA1), 2; darker than control...sequence the resulting plasmid. It can take roughly 3 months from the day you decide to do a gene modification...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...

...Topic Page Learn about Non-Homologous End Joining Learn about Base Editing Read 3 Tips to Improve ... break is resected by nucleases to create a ssDNA 3’ overhang. This will serve as both a substrate for... repair (DSBR) In the classical DSBR pathway, the 3’ ends invade an intact homologous template to serve...the non-invading strand at the other DSB end. The 3’ end of the non-invasive strand is elongated and ligated... donor DNA. Richardson CD, et al. Nat Biotech. 34(3); (2016). PubMed. Efficient homology-directed repair...

...the CEO of 3-D Molecular Designs.     Additional Resources on the Addgene Blog Learn All about CRISPR Use...MSOE center for BioMolecular Modeling and the CEO of 3-D Molecular Designs. Have you ever held your favorite...models were useful in that they not only traced the 3-D path of the protein’s backbone in three-dimensional...arginine 53 really is right there, at the end of helix 3…just like we said it was”.   Seeing is believing. ...Until then, you can only wonder what I am talking about. How do researchers use physical models of protein...students work closely with local researchers to learn about their research while creating physical models of...model is an effective teaching tool. You can read about Jacob’s initial reaction to this model on his blog...

...field, (2) that you have three 3 years of research experience in the field, (3) that you have an offer of ...applications and approved 91,023 during the first 3 quarters of 2017. As an international researcher and...well positioned to advance the proposed endeavor; (3) The foreign national’s proposed endeavor will be ...for this category. To qualify, you need to satisfy 3 out of the 10 criteria listed below. An international...of the institute. For the EB-1B, you need to prove 3 threshold requirements: (1) that you have international... you must show that your company employs at least 3 full-time people engaged in researcher activities.... data and the list goes on and on. As you think about your career (and your future in general), keep in...

...three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required for recombineering with...DNA introduced into the E. coli. Exo: Exo is a 5’→3’ dsDNA-dependent exonuclease.  Exo will degrade linear... 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the dsDNA was short enough, a ...the translated protein.  These changes can be 5’ or 3’ of the desired change.   Table 1: Overview of ds-... integrated defective prophage 2) from a plasmid, 3) from mini-λ or 4) from the lambda red phage itself...PubMed PMID: 10811905.PubMed Central PMCID: PMC18544. 3. Murphy, K. 2016. λ Recombination and Recombineering...currently a research scientist in the Department of Laboratory Medicine at the University of Washington. She...

...mutations in RPE65 (n=3), showed significant improvements in vision over a period of 1 to 3 years – the treatment...neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are thought to be as high as 70% among the human...PMID: 20463753. PubMed Central PMCID: PMC3152950. 3. Jacobson, Samuel G., et al. "Improvement and decline...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.     ...

...15;8(3):535-42. PubMed PMID: 23256573. Additional Resources on the Addgene Blog Learn More About the ...yeast as an alternative source of essential omega-3 fatty acids complementing vegetable oil, a somewhat...PMID: 27984002. 2. Bilan, Dmitry S. et al. “HyPer-3: A Genetically Encoded H2O2 Probe with Improved Performance...The Addgene-Seeding Labs plasmid grant is a collaborative endeavor providing plasmids to researchers in... of Zimbabwe, Zimbabwe Dr. Idah Sithole-Niang (above, left) is an Full Professor in Biochemistry with... CRISPR effect observed in bacteria. Learn more about the different CRISPR applications in plants.     ...Rodrigo Arturo Rivera Solís, Associate Professor (above, far right), thrives to make affordable biosensors...

...Figure 1, large sequences corresponding to exons 1 and 3 of the targeted gene have been cloned into the vector...Neomycin resistance (NeoR) gene between exon 1 and 3 of the targeted gene. Upon recombination, the GFP/...-PGK-Puro from the Jaenisch lab.   Figure 3: A knockout/knock-in targeting vector designed to ...resistance marker using Cre recombinase. Since there are 3 loxP sites, recombination can occur in multiple ways...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  ...

...sequence (Zong et al., 2018) (see CBE-3 in Figure 3). Figure 3: Activity windows of plant base editors...position 4-8 counting the ‘NGG’ PAM as 21-23 (Figure 3). If the target ‘A’ is closer to the PAM, i.e., positioned... 353.6305 (2016): aaf8729. PubMed PMID: 27492474. 3. Gaudelli, Nicole M., et al. "Programmable base editing...gene, Xw, except for a single base. If you heard about CRISPR, one question probably comes to mind: can...compatibility All of the base editors mentioned above are SpCas9 nickase-based and depend on the presence... HDR methods for rice crop improvement in the laboratory of Yinong Yang at PSU.   References 1. Komor,...The CRISPR-Cas Revolution, Cold Spring Harbor Laboratory (2018). Additional resources on the Addgene blog...

...more than 3-4 colors due to the tandem integration of multiple copies of the construct (about 8 in Brainbow...colorful technology. Optimizing the system: Brainbow-3 Although Brainbow provided neuroscientists with the...Looger LL, Simpson JH. Nature Methods. 2011 Mar;8(3):253-9. doi: 10.1038/nmeth.1566. Epub 2011 Feb 6. ...Dickson BJ, Salecker I. Nature Methods. 2011 Mar;8(3):260-6. doi: 10.1038/nmeth.1567. Epub 2011 Feb 6. ...Beaurepaire E, Morin X, Livet J. Neuron. 2014 Feb 5;81(3):505-20. PubMed.   ...with the goal of overcoming the limitations listed above. First, they screened a variety of fluorescent proteins...method has become very useful in lineage tracing; “Zebrabow” was used to trace the development of the corneal...

...in plants. When expressed, the construct produces 3 enzymes from a single mRNA using 2a sites. These convert...Betacyanin is produced from tyrosine by the activity of 3 enzymes and a spontaneous step. (B) RUBY is a reporter...collect seed for further experiments.     Fig. 3: (Top right) A RUBY-expressing Arabidopsis seed following... is difficult for new members of the lab or collaborators unfamiliar with these mutant plants. Using RUBY...resistant disease. (RUBY-red: You can’t beet it!) About the Author  Andrew Willoughby is a graduate student...