...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...

...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...

... 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...

...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...

...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. ...

...., 2022), indicating that there is more to learn about this process!      Figure 3: Crystal structure...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...

...included in a publication, you'll find that information in the report as well.    Figure 3: An antibody...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 ...very little data on which might work in your application. It can be quite difficult to decide on which...be searched and sorted by gene/target, name, application, pass/fail rating, sample species, antibody species... how the antibody performed in the submitted application, along with a brief description of the experiment... section will vary slightly depending on the application — flow cytometry, western blot, immunohistochemistry...

...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:...

... as their orientation relative to the gRNA (5’ or 3’). In some cases, there is no requirement, which is... spCas9 isn’t always the winner!).   Fig. 3 – Comparison of different Cas enzymes using CasPEDIA...established uses, and intended applications. Within CasPEDIA, there is a classification system, CasID, to help...multiplex ability Experimental guidelines and applications Protein structure and properties Links to ...you want to read literature on the less common applications of an enzyme for your non-traditional experiment...al. (2023). CasPEDIA Database: A Functional Classification System for Class 2 CRISPR-Cas Enzymes [Preprint...I., Iranzo, J., et al. (2019). Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and...

...Seeger lab designed 3 different mRNA sybody libraries for use in the protocol, which cover 3 naturally-occurring...biotinylated (for purification purposes) protein target of interest, the protocol goes through 3 major steps... against membrane protein targets in as little as 3 weeks (Zimmermann et al., 2020). This selection platform...structures (concave, loop, and convex). Each of these 3 mRNA libraries have a concave, loop, or convex scaffold...-30) of sybody candidates are sequenced for identification. All unique sybodies can be subcloned into ...2020) Generation of synthetic nanobodies against delicate proteins. Nature Protocols 15:1707–1741. https...Light to Manipulate Cell Signaling and Protein Purification RANbodies: Reporter Nanobody Fusions Plasmids...

... 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...equivalent to backbone + insert, unsuccessful 2 and 3 above), you can try ligating the recipient plasmid...colonies and check them for successful ligation. Pick 3-10 colonies depending on the number of background ...them. An easy way to do this is gel purification. In gel purification, you use a voltage difference across...

...HSPG)3 AAV receptor (not strictly required)2 Human fibroblast growth factor receptor 1 (FGFR1)3 αVβ5...Hepatocyte growth factor receptor (HGFR)3 Laminin receptor (LR)3 CD93 AAV5 2,3-N-linked sialic acid4... (PDGFR) α and β3 AAV8 Laminin receptor (LR)3 AAV receptor2   AAV9 Terminal N-linked galactose3...galactose3 AAV receptor2 Laminin receptor (LR)3 Putative integrin3 1Wu, Miller, et al., 20062Pillay...Journal of the American Society of Gene Therapy, 14(3), 316–327. https://doi.org/10.1016/j.ymthe.2006.05.009...vessel barriers. It is especially popular for applications targeting liver tissue. In addition, it has ...the blood-brain barrier, making it useful for applications targeting the CNS (Issa et al., 2023). Additionally...

...sequences at its 3’ and 5’ ends that are crucial cis-acting signals for genome replication and viral gene...genome. It contains the 3’ and 5’ ends of the genome which are required for replication and transcription (...synthesis at the 3’ end of the minigenome to produce the positive sense, genome replicative intermediate ... PMID:15298166. 3. Hoenen, Thomas, et al. "Minigenomes, transcription and replication competent virus-...nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola virus...Technical Tips). Minigenome replication and transcription In order to replicate the minigenome, the viral...transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems...