...PMID: 27347757. Find plasmids from this publication at Addgene. 3. Zetsche B, Gootenberg JS, Abudayyeh ...01200-3. doi: 10.1016/j.cell.2015.09.038. PubMed PMID: 26422227 Find plasmids from this publication at ...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...

...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...databases available, Web of Science. For each publication, Web of Science provides basic bibliographic ...Science, PLOS One, and PNAS. We then broke the publications from these journals into two groups: those containing...

...-TTN, differing from that of Cas9 (3’-NGG) in both genomic location and GC-content. After sequencing and...PMID: 27918548. Find plasmids from this publication at Addgene. 3. Kim, Daesik, et al. Genome-wide analysis...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. Cpf1’s strength may ...

...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 ... While ribosome profiling co-opts the physical location of the ribosome, TRICK (Translating RNA Imaging...development, the osk-TRICK reporter was double labeled, indicating translational repression. During later stages...ribosomes after initiation, to calculate a ribosome translocation rate of ~3.5 codons/second. They also explored...

...spending time with her horse, and wondering what her cat is thinking. Find her on Twitter @JessicaSacher. ... 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...

...2018 Jun 14;11(1):387. doi: 10.1186/s13104-018-3486-3. PubMed . Promoter-associated proteins of EPAS1 identified...of a catalytically inactive form of Cas9 (dCas9) plus guide RNA (gRNA) for biochemical purification (Fig...protein such as LexA are subjected to affinity purification (Fig. 1). In enChIP, specific genomic regions...537-548. doi: 10.1093/dnares/dsx023. PubMed . Identification of physical interactions between genomic regions...10.1111/gtc.12341. Epub 2016 Feb 5. PubMed . Identification of non-coding RNAs associated with telomeres...26. doi: 10.1186/s12867-014-0026-0. PubMed . Identification of proteins associated with an IFNgamma-responsive...Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding...

...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...to the presence of additional helper sequences (2-3). As alluded to above, termination and polyadenylation...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 ...type are generally broken down into two broad categories, prokaryotic or eukaryotic, based on the functional...

...Gutless AdV can be found in reference 3. Production, amplification, and quality control of 2nd generation...size, and consists of an icosahedral capsid made of 3 types of proteins: fiber, penton and hexon proteins...sequenced to confirm its integrity. Initial production (2-3 weeks) - Here you’ll produce the primary recombinant... will turn yellow. Do NOT change the media (add 2-3 mL of fresh media once a week), and do NOT harvest...infected cells will become round and clump together ~3 days post infection. The amplified virus can be harvested...al. A practical guide for using the AdEasy System. 3. Jager, Lorenz, et al. "A rapid protocol for construction...Each round of amplification should result in a 10-100-fold increase in virus. Purification (2 days) - Purification...

...between deletion size and frequency [3]. There are several applications in which the production of deletions...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... of Visualized Experiments (JOVE). The video publication by Stuart Orkin and Daniel Bauer's labs details...useful to study haploinsufficiency. Subsequent modifications at the remaining allele within the deletion ...

... 53 potential candidate genes that fell into 3 categories based on the architecture of the CRISPR protein...analogous to the PAM sequence for Cas9, is located at the 3’ end of the spacer sequence and consists of...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...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...use, functional in most species, and has many applications. That said, SpCas9 is not the only game in town...

... 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... Specifically, viruses are able to infect and replicate in host cells, but they are susceptible to cells... though not all, of these defenses. For some applications of genome engineering - such as certain forms...to integrate. Additionally, the TA sequence is duplicated near the gene of interest insertion site.   The...1) an SB transposase, the enzyme required for catalysis of transposition; and 2) a transposon containing...

...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...radioactivity in the culture supernatant (this is indicative of protein degradation). To control for protein...studying autophagy. This method relies on the identification of autophagic structures based on morphology...

... or not you’ve succeeded in communicating the information you intended. 3. Join the scicomm community ... who are passionate about communicating science.  ComSciCon's events have 3 main goals: To help graduate... the Communicating Science Conference series for graduate students. I believe that communication is the...skill in science communication over time.  Like most skills, great science communication comes with practice...able to communicate with their colleagues and peers. Scientists also need to be able to communicate to expert...need to communicate with non-scientists, to establish broader impacts for their work in education and policy...with opportunities to practice communicating.  Some fantastic K12 education initiatives like BiteScis (https...

...expression was across the different cells (Figure 3).  3. In terms of making the monoclonal line: I had ...thing ask incredulously, “You seriously didn’t add 3 microliters of 5 mM star anise?” This is something...would still be able to identify the cleavage product. 3. The Surveyor kit had a lot of good tips for first...engineering has come. For comparison, in 2007 it took about 3 months to knockout a single allele in a human line... BL, Xavier RJ, Root DE. Nat Biotechnol. 2014 Sep 3. doi: 10.1038/nbt.3026. 10.1038/nbt.3026 PubMed PMID...PMID: 25184501. PubMed Central PMCID: PMC4262738. 3. EGFP gRNA (BRDN0000561167) (Plasmid #80034) and BRAF... wanted to inactivate the gene BRAF (a kinase implicated in several human cancers) in A549 cells (a human...

...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.,... OriP and induces amplification of the episomes concurrently with DNA amplification in the host cell. ...

...shRNA cassettes into the unique SnaBI site in the 3´-LTR). See here for other versions of pULTRA. Moore...included to monitor expression Parijs 11619 pLB 3rd Modification of pLL3.7; Genetic elements known to prevent...

...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... (AmpR) and origins of replication (ori and f1 ori) which allow it to replicate.   How can I prevent...the bacterial selection marker and origin of replication. During bacterial culture, only the plasmid with...the antibiotic resistance gene and origin will replicate, and the transgene will be lost. Over time, bacteria...the recombined plasmid is still visible. This indicates that the DNA prep contains a mixture of the full...

...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? ...that have been used during the purification procedure. Good indicators of DNA purity are the absorbance...nm should fall ~1.8 - 2 and give scientists an indication of purity versus protein contaminants. The 260...nm ratios should fall ~2 - 2.2, and provide an indication of purity versus chaotropic agents as guanidine...

...which cell type? Location (3): Traveling from the original location - Axonal Transport in the CNS What...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 ...

...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...additional origins of replication compared to monomers, they are also able to replicate at a higher frequency...expected fragments for that plasmid. This phenomenon indicates the presence of a multimer.   Fig. 1:...