... 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) ...

...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...

... 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...

...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...

...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...

...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...

...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...

...): 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...