We narrowed to 735 results for: RAN-1

...references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid is identified, re-transform into a bacterial...rAdV-S). HEK293 cells are transfected with the AdV plasmid construct from step #1 and allowed to stay in ... (ITR) present at both ends.   Figure 1: A) The structure of an adenovirus: the virion is ~ 100nm... 5 key steps: Construction of the rAdV plasmid (~1 week): The AdEasyTM system is the most popular method... it will result in very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more HEK293...-4 rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification should result...CsCl are commercially available. Titer calculation (1 or 10 days) Physical titer (particle count, rAdV ...

... Oligos Oligo 1: Add 'tcaa' to the 5′ end of your forward guide sequence (see Figure 1). Oligo 2: Add... Figure 1). Perform standard phosphorylation and annealing of the two oligos. Figure 1: Components... promoter for ωRNA (reRNA) expression (see Figure 1). Both plasmids contain a hygromycin resistance gene...provide a step-by-step guide for using a Dra2TnpB. Step 1: Design the 20-nt Guide Sequence Identify a TAM site...protocols (pp. 245–260). https://doi.org/10.1007/978-1-0716-4358-7_20  Karmakar, S., Panda, D., Panda, S... induction of meristems. Nature Biotechnology, 38(1), 84–89. https://doi.org/10.1038/s41587-019-0337-2...1166–1187. https://doi.org/10.1038/s41477-021-00991-1  Qiao, J., Zang, Y., Gao, Q., Liu, S., Zhang, X.,...

.... "Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and...Examining serotypes 1, 8 and 9, not only do they undergo retrograde and anterograde transport, but they also...serotypes 1, 8, and 9 share conserved mechanisms for anterograde and retrograde axonal transport." Human...vectors without specific facilities (Biosafety Level-1). Other possible tools, lentiviruses for example, ...suspected cases of insertional mutagenesis in humans (1) and AAVs are non-enveloped viruses, so they are resistant...Subscribe to Viral Vector Blog Posts from Addgene 1. Location, location, location! What do buying real...important factors related to location. Location (1): What tissue? Location (2): Where in the tissue/which...

...intracellular targets. Nature Communications, 7(1), Article 1. https://doi.org/10.1038/ncomms10778  Erdmann...small molecules in vivo. Nature Biotechnology, 21(1), 1. https://doi.org/10.1038/nbt765  Lavis, L. (2016... envelope design. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023-40788-8  ... capping protein. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023-39655-3  ...up linked to whatever molecule you choose (Figure 1). Just express the fusion protein and add a specific...proteins, opening up new applications.   Figure 1: Live-cell labeling strategies. The green structure...minimally-disruptive genetic tag on the protein of interest. Table 1: Summary of popular self-labeling tag systems ...

...can use Figure 1 to compare the genomes of these two family members.   Figure 1: Gamma-retrovirus...Retroviruses” is an umbrella term which encompasses 1.) the gamma-retroviruses — that have been developed...of retroviruses called the lentiviruses (like HIV-1) — that have been developed into lentiviral vectors...Academy of Sciences of the United States of America, 54(1), 137–144. https://doi.org/10.1073/PNAS.54.1.137. ...1997). Functional RT and IN incorporated into HIV-1 particles independently of the Gag/Pol precursor protein...RNA as template. Journal of Molecular Biology, 79(1), 163–183. https://doi.org/10.1016/0022-2836(73)90277...Aids Full-Length Human Immunodeficiency Virus Type 1 DNA Synthesis. Journal of Virology, 82(5), 2376. https...

...HIV-1 were transduced with a lentiviral vector containing an antisense sequence against the HIV-1 envelope...infect. Finally, the transfer plasmid encoded the desired transgene flanked by HIV-1 long terminal repeats...packaging, envelope, and transfer plasmids. The packaging plasmid carried a mutated HIV-1 provirus that couldn...section below! References 1. In vivo gene delivery and stable transduction of nondividing cells by a ...Trono and collaborators turned to the lentivirus HIV-1, well-known to infect non-dividing cells. The first... targeting and tracing cells in the brain (Figure 1). 2nd generation lentiviral packaging system The graphic...infectivity, is often used as a substitute for HIV-1 Env, which can only infect CD4+ cells. 3rd generation...

...Cascade's complexity. Figure 1: Schematic of Class 1 CRISPR types. Class 1 includes type I with a Cas3 ...collection! A "complex" system Cas3 is part of the Class 1 CRISPR family, which means it requires multiple Cas...the brunt of the mechanical work for editing. Class 1 CRISPR systems make up the majority of CRISPR-based...CRISPR-based immune systems in bacteria and archaea. Class 1 is further broken down into different types, with ...making up type I, the most abundant type in the Class 1 category. These type Is can then be further broken...editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-13226-x Cameron...non-target strand. The helicase domain is then activated, and Cas3 begins unraveling the double-stranded DNA ...

...Figure 1).   Figure 1: Each problem with dimerizing fluorescent proteins is illustrated. 1) Membrane... dimerization.   1. Transmembrane fluorescent protein fusions Transmembrane proteins, i.e. receptors... in nature are prone to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [...bright, non-physiological, and difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several... For a more detailed discussion see [5]. Problems 1 and 2 depend on protein concentrations. An imperfect... distance runner, gardner, and cook.   References 1. Snapp, E.L., et al., Formation of stacked ER cisternae...contacts that bring apposing membranes together and can wrap the membranes into stacked structures including...

...their HLDA workshops. Let’s use TIM-1 (Single pass type-1 membrane protein), which has been designated...dye, respectively. WT and KO cells were mixed in a 1:1 ratio and fixed in 4% PFA and permeabilized in 0.1%...antibodies were diluted to 1 µg/mL except for 14558, which was used at 0.35 µg/mL (1/100). * = monoclonal antibody...specific antibody:target combination of interest (Figure 1). It can therefore be helpful to try all three when...to label an intracellular target.   Figure 1: HCT 116 WT and SYT1 KO cells were labelled with a...cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb CoraLite® Plus...between the wild-type and the KO population (Figure 1), further optimization can be performed to both maximize...

...approximately 1 in 16 mice that harbor homozygous alleles at both of your genes of interest. About 1 in 4 mice...ve learned in Part 1, there are many types of genetically engineered mice: transgenic mice, knockin and...In Part 1 of our mouse modeling blog series, we covered techniques that can be used to introduce genetic..., and assuming Mendalian patterns of inheritance: 1. Genotype your parental mice In order to ensure that...example of mice that are derived from crosses. In Part 1, we explained how genetic recombination in mice expressing...expressed, or inducible (this is explained more in Part 1 of this series). In any case, Cre is a dominant phenotype... integration of a novel gene randomly into their genome, some transgenic strains are difficult to breed...

...enzyme (among others): caveolin 1, also known as CAV 1. Usually, caveolin 1 promotes the cell life cycle ...caveolin 1, there’s less malignancy because there’s less proliferation. However, the lack of caveolin 1 in ... expression of TAK 1, an enzyme that controls apoptosis and regulates transcription. Without TKA1, cells...Agnieszka K., et al. "An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor...microenvironment. This results in increased expression of transforming growth factor-β signaling, possibly inducing...let’s take a look at exosomes. Proficient in transportation, exosomes are tools of communication and provide... (micro RNA) and other molecules that can be transferred to other cells, inducing epigenetic changes in...

...and drawbacks of four split AAV vector approaches. 1. Overlapping  Overview: The overlapping strategy has...Adeno-Associated Virus Capsids. Human Gene Therapy Methods 27:1–12 . https://doi.org/10.1089/hgtb.2015.140 Frischmeyer... successfully mediated transduction and expression of the full-length transgene in vitro and in vivo (...this, larger functional transgene products were produced following transduction.  So what’s going on here...between part A and B of the transgene to reconstitute the full-length transgene (Chamberlain et al., 2016...cells in mice. This vector transduced ~10% of cells, similar to the transduction rate of a single AAV. Odom...muscle physiological performance.  2. Trans-splicing Overview: For trans-plicing, splice site donor and acceptor...

...nucleotides 1-10 of the guide RNA, prearranged for base pairing with a complementary DNA strand. These 10...4CMP) with domains colored as in Figure 1. HNH, red; RuvC, orange; CTD, yellow; REC lobe, light blue; bridge...10.1146/annurev-biophys-062215-010822 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I.... basic parts of the Cas9 enzyme.   Figure 1: A cartoon depiction of Cas9’s two major lobes, REC...the CRISPR system (Nishimasu et al., 2014). Table 1. Summary of Cas9 domains, the lobe each domain belongs... on itself at the bottom of the image. Stem loops 1 and 2 extend horizontally across both lobes. The guide...outside of the protein, spanning both lobes. Stem loop 1, nestled between the REC and CTD domains, is required...

...and no viral progeny are produced (Figure 1).  Figure 1: Comparison of the life cycle of a lentiviral...2004). HIV-1 gene expression: lessons from provirus and non-integrated DNA. Retrovirology, 1, 13. https... cannot integrate it into the host genome (Figure 1). IDLVs were first discovered by researchers investigating...you might choose an IDLV over a lentiviral vector: 1. No risk of insertional mutagenesis Lentiviral vectors..., H. E. (1996). Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity...Berry, C., Ecker, J. R., & Bushman, F. (2002). HIV-1 integration in the human genome favors active genes...https://doi.org/10.1186/1742-4690-1-13. PubMed PMID: 15219234 PubMed Central PMCID: PMC449739 Yew, C.T., ...

...vectors. BMC Biotechnology, 6(1). https://doi.org/10.1186/1472-6750-6-1 O’Brien, J., Hayder, H., Zayed...machinery for processing (Figure 1). The shRNA sequence is transcribed to produce hairpin shRNA structures... recruits other silencing proteins. Figure 1: Comparison of shRNA and miRNA processing and silencing...,"rgb":"rgb(0, 82, 169)","rgba":"rgba(0, 82, 169, 1)"},"button_font":{"size":19,"styles":{"font-family...:"rgb(255, 255, 255)","rgba":"rgba(255, 255, 255, 1)"}}{% endraw %}{% end_module_attribute %}{% module_attribute...RNA-induced silencing complex. Molecular Cell, 82(1), 30–43. https://doi.org/10.1016/j.molcel.2021.11.026...progress in miRNA biogenesis and decay. RNA Biology, 21(1), 36–43. https://doi.org/10.1080/15476286.2023.2288741...

...of the desired T-A base pair. Figure 1: Transition mutation (C>T, G>A, A>G, and T>C) installation...hAPOBEC3 deaminase can edit in a stretched window ranging from 1-17 bp relative to the PAM sequence (Zong et...excision repair mediated by uracil glycosylase (Figure 1). The presence of a uracil glycosylase inhibitor (... A-T base pair is converted to a G-C pair (Figure 1). As the cellular inosine excision repair is not as...Target-AID based (CBE-2)-  and rAPOBEC based (CBE-1)-  CBEs are successful base editors in rice, tomato...which recognize PAM sequences other than NGG (Table-1). Type Nickase Cas9 (D10A) Deaminase PAM requirements...locus/loci, choose a base editor variant from Table 1 depending on the PAM sequence. Avoid targeting a ‘...

...sensing glutamate using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation and ...hSynap: Human Synapsin-1 promoter. Good for neuronal expression. hSynap-FLEX: Synapsin-1 promoter, FLEXed for... v857. Image adapted from Aggarwal et al., Figure 1   Increased versatility through two different...expression in Cre-expressing cells. hSynap-FLP: Synapsin-1 promoter, FLP-dependent expression GFAP: Glial fibrillary...three different membrane-targeting flavors – PDGFR, GPI, and SGZ, to improve neuronal membrane trafficking...properties such as kinetics, ∆F/F0, SNR, dynamic range, photostability, quantum yield, and expression. ...segment (PDGFR TMD) that anchors the sensor to the membrane and displays it on the extracellular side. In ...

... Eric Campeau 21474 pLenti CMV V5-LUC Blast (w567-1) Firefly CMV Lentiviral expression of firefly luciferase...Bioluminescence Resonance Energy Transfer (BRET) Similar to FRET, energy transfer from the luciferase reaction...can be used to study processes beyond transcription and translation. Splicing reporters from Younis et al...bioluminescence found in a variety of organisms, ranging from bacteria to aquatic animals to insects. Luciferase...photons. Its robust signal, sensitivity, wide dynamic range, and resistance to photobleaching have made luciferase...Regulatory elements such as promoters, enhancers and untranslated regions, or shRNA or CRISPR target sites can... protein half-lives, making them useful as transcriptional reporters, but are less-than-ideal protein ...

... Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from Li et...coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella citrea, with a total of 70 sites...the pCas/pTargetF system.   Figure 1: The editing process begins with the addition of the sgRNA sequence...updates involved the following changes. In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2)...biosynthesis, we constructed a strain library with 1-11 copies of integrated genes. We obtained a stable...in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF into the BL21...pTnsABC expresses the transposases TnsA, TnsB, and TnsC pQCascade expresses the transposase TniQ, the Cas proteins...

...Davidson AR.  Nature. 2013 Jan; 493, 429–432. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...sequences were initially discovered in E. coli in 1987 (1), the concept that these clustered repeat sequences...neutralize CRISPR/Cas. Earlier this year, expression of 1 of 5 anti-CRISPR genes were found to inactivate the...and are not translatable to the other CRISPR subtypes. Still, the large and dynamic range of CRISPR systems...well-suited for various innovative biological and translative technologies. Addgene has empowered researchers...http://en.wikipedia.org/wiki/File:Crispr.png Barrangou R, Horvath P. Science. 315, 2007 Jan; 1709–1712...CRISPRdb. Date: 2013-09-18. Makarova K, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, Moineu...

...Biotechnology, 10.1038/s41587-023-01756-1. https://doi.org/10.1038/s41587-023-01756-1. PMID: 37095348. Breaking... for molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038...incubation, even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused to HIV TAT, Myc and...expressing COX8a-mStayGold. Scale bars 10 μm (main), 1 μm (inset). Image reused from Ando et al. 2023, under...StayGold fluorescent protein. Nature Biotechnology, 1–4. https://doi.org/10.1038/s41587-023-02018-w. PMID...broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467-023-38582-7...StayGold variants AAV.CAP-Mac for systemic gene transfer in primate brains Endothelial-specific AAV delivery...

...successfully identified using this method. Step 1: Prepare a random fragment library of phage DNA This first ...and leave for 1 h at room temperature. After this, it is helpful to subject your membrane to several freeze... the membrane itself or to any “sticky” proteins expressed by your library. After blocking for 1 h at ...it can really help to incubate the membranes overnight in 500 mM to 1 M NaCl in order to enhance folding...place to start) and add your membranes to the solution, shaking gently for 1 h at room temperature. This... on Twitter @JessicaSacher.   References 1. Nobrega, Franklin L., et al. "Revisiting phage therapy: new...which is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...

... PCR leads to the most colonies after transformation. When a 1:1 vector:insert ratio is used in the DpnI... get tips for applying it in your own lab. Table 1: Comparison Between Standard Restriction Cloning and...Connecticut interested in protein science.     References: 1. Celie, Patrick HN, Annabel HA Parret, and Anastassis...PCR cloning method." BMC biotechnology 11.1 (2011): 1. PubMed PMID: 21992524. PubMed Central PMCID: PMC3207894...products are then transformed directly into chemically competent E. coli and transformants selected with ... for further purification before transformation. After transforming cells, you should perform colony ...consisting of digestion, purification, ligation, and transformation. While restriction cloning is still routinely...

...marker. Looking at Table 1, we can recognize a couple of fluorophores from Figure 1. For example, the chart...called Fluorescence Resonance Energy Transfer (FRET).   Figure 1: Emission spectra and respective ...efficient (i.e., happen most of the time). Figure 1 shows a graph from the BD® Spectrum Viewer, outlining... the brightness of individual fluorophores. Table 1 describes the brightness of a few commonly used fluorophores...Brighter fluorophores have a stronger signal. Table 1: Degrees of brightness for a few commonly used fluorophores...blue laser excites at a wavelength of 488 nm (Table 1), which is near a local maximum of PE's excitation...Knowing that the red laser excites at 640 nm (Table 1), while comparing the two fluorophore's excitation...

...of publication. Table 1 provides an example calculation of the weights. Table 1: Journal Weighting Example...amassed nearly 2,400 citations in its first four years (1). In addition to publishing in Science, Professor ...papers without plasmids deposited with Addgene (Figure 1). Even aggregated this much, you can see the 2013...(F) Weight (=F/E) (G) Weighted Citations (=C*G) 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010...website at: http://www.neil-t.com/ .   References 1. Cong, Le, et al. "Multiplex genome engineering using...on a range of topics including biotech and research tools, computing performance, and randomized control...

...aggregation, and potentiates the effect of PD-1 blockade.“ (Ref 4). PD-1 helps tumor cells evade the immune system...fasting has a positive effect on cancer treatment (Ref 1). As described in the presentation, starvation or ...of the immunosuppressive cell surface receptor PD-1. This immunosuppressive environment in turn may represent...  (Chimeric Antigen Receptors) CAR T cells and PD-1 modulators. An exciting development in immunotherapy...free deposit and distribution service! References: 1. Pietrocola, Federico, et al. "Caloric restriction...discussed and Professor Caetano Reis e Sousa from the Francis Crick Institute concluded “Aspirin promotes autophagy... Kettering Cancer Center. In contrast to using randomly integrating viral vectors, CRISPR/Cas9 genome ...

...-Integrin alpha 5 beta 1 [IPI-ITGA5/ITGB1.2] Integrin alpha-5,Integrin beta-1 Human, Mouse IgG1 Rabbit...-Integrin alpha 5 beta 1 [IPI-ITGA5/ITGB1.4] Integrin alpha-5,Integrin beta-1 Human, Mouse IgG1 Rabbit...specific integrins in cellular processes.   Figure 1: Integrins can shape into three conformations: (A)...immunoprecipitation and immunohistochemistry. Table 1: IPI antibodies  Antibody Target Proteins Reactivity...Human IgG1 Rabbit Anti-Integrin alpha M [IPI-LM2/1] Integrin alpha-M Human IgG1 Rabbit Anti-Integrin...Human IgG2a Mouse Anti-Integrin alpha M [IPI-LM2/1] Integrin alpha-M Human IgG2a Mouse Anti-Integrin... β1 integrin mechanics. Nature Communications, 13(1), 7471. https://doi.org/10.1038/s41467-022-35173-w...

...between the cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase I (1). If a 5′ hydroxyl group ...plating your transformation may allow you to see colonies within 8 hours. References: 1. Shuman S. "...Let’s break down the steps needed for TOPO cloning: 1. Create Your PCR Product: Design standard primers ...mind that Taq polymerase has an error rate of about 1 in 3,500 bases. Typically polymerases with proofreading...enzyme and Taq, with Taq used in an excess ratio of 10:1. Gel purify your PCR product and incubate it with ...different DNA strand comes along, it can attack this covalent bond thus joining the two DNA strands and releasing...planning to transform right away OR you can store the reaction at -20C overnight. 4. Transform TOPO Cloning...

...need a job. Start now. Informational interviews are 1:1 discussions with someone who has interesting experiences...conversation builds a relationship for the future and the 1:1 format is good for everyone including those that ...out-of-the-box ideas that might jump start your learning. 1. Create a peer mentoring group We don’t all get the... and not in academia is run by grant funding. Learning to write grants quickly, efficiently and effectively...apply to or offer to help with lab grant writing. You may not get the grant or your PI may edit everything...thinking about future career options. 5. Write a grant - even if you don’t need one A lot of science in...valuable skill. There are many different kinds of grants that you can apply for. If you are in a lab, your...

...estimated to affect anywhere from 1 in 6,000 to 1 in 19,000 live births (1).  While disruption of the smn1... funding opportunities. References 1. Jones, C., et al. "PP09. 1–2352: Systematic review of incidence...disease. This year, Addgene will be providing plasmid grants worth $2,000 each to two labs through the BeHEARD...

...spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer team will communicate...scientist, you will have the most involvement with Step 1, and, while data entry can be a chore, we’ve made ...Deposit” and “Download Deposit Spreadsheet” (see Figure 1). Please read our “How to Deposit Your Plasmids with...thorough guide to our online data entry process. Figure 1: Begin the deposit process by clicking “Submit Online...spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down options for you to choose...and are exceptions to our standard workflow.  Table 1: Guidelines for the data fields in the deposit spreadsheet... Send plasmid information to Addgene Material Transfer Agreement (MTA) approval  Send plasmids to Addgene...

...type, and/or desired event.   Figure 1: Transduction to expression process. A viral particle enters...triple T’s of biology are transformations, transfections, and transductions! In this blog we will discuss...intricacies of transductions, we've got you covered there too. What is a transduction?  Transduction is the ... experiment. Using transduction vs. another method Transfection and transduction seem to achieve similar...picky when it comes to transfection/transduction methods. Some ‘difficult to transfect’ cells lines are amenable... process of transferring foreign nucleic acid into a cell by a viral agent. Transductions are similar ...to find out more! Molecular basis of transduction Transductions utilize the ‘normal’ mechanism of viral...

...Jacqueline N Crawley  [et al]. 2012;Chapter 1:Unit 1 20 1-18. doi: 10.1002/0471142301.ns0120s59. PMID:...mutually exclusive) commonly used in optogenetics: 1) electrophysiology and 2) behavioral experiments. ...can be broken down broadly into three main steps: 1) Selection and expression of your opsin of choice ...reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn about...monitor expression at various time points (e.g. every 1-2 weeks over 8 weeks) in order to determine the optimal...follow him on Twitter @derekpsimonphd.    References 1. Tye KM, Deisseroth K. Optogenetic investigation of..., Jacqueline N Crawley  [et al]. 2013;4(435):4 35 1-4  20. doi: 10.1002/0471142301.ns0435s65. PMID: 25429312...

...near-perfect physical-to-infectious particle ratio of 1:1 (Zeltner et al., 2010). However, for recombinant ...preparations are genome-containing (Figure 1).    Figure 1: Electron micrograph of an Addgene AAV...reported a physical-to-infectious particle ratio of 50:1 (Zeltner et al., 2010). The specific infectivity of... lot (if we still have it available). References 1. Fagone, Paolo, et al. "Systemic errors in quantitative... Human Gene Therapy, Part B: Methods 23.1 (2011): 1-7. PubMed PMID: 22428975. PubMed Central PMCID: PMC3640491...particles that can transduce cells. Infectious titers are typically quantified by cell transduction assays. Wild-type....qPCR...Digital droplet PCR...DNA dot blot...Transduction assay...SDS-PAGE...Electron microscopy… Any ...

... for studying translation in various cell types and physiological states. References 1. Ingolia, Nicholas...various stimuli on global translation, but a visual technique to study translation remained elusive. Two ...easier to track translation in two different ways: by monitoring the first round of translation or by tracking...destination co- or post-translationally. TRICK: Visualizing the first round of translation While ribosome profiling...in the fluorescent signal: untranslated RNAs appear yellow, and translated RNAs appear red. To enable ...Using translation inhibitors cycloheximide and puromycin, Halstead et al. verified that translation is necessary...readout of relative reporter translation. Single molecule continuous translation tracking TRICK allows you...

...approximately 20 nm of a specific DNA sequence in only 1 min. Using these lentiviral plasmids, catalytically...expressing a novel reverse transcriptase (RT), MarathonRT. Reverse transcriptases are critical tools for ... to transform and express in bacteria, which, in turn, makes them more adaptable to a broad range of applications...!  MarathonRT: A highly processive reverse transcriptase Article contributed by Will Arnold  Listen ...RTs in this regard and is even able to reverse transcribe the extremely long, highly structured HCV genome...structured template!). MarathonRT also faithfully transcribes RNA to DNA with low error rates compared to other... are dependent on long, error-free, reverse transcription of RNA such as long-read cDNA sequencing and...

...deletions, point mutations PCR Reactions 1 1 1 per fragment 1 per fragment In vitro Assembly No No Yes...methods for site directed mutagenesis   References 1. Trehan, Ashutosh, et al. (2016). “REPLACR-mutagenesis... and the nonmethylated PCR products are then transformed into E. coli to undergo nick repair. This method...containing the desired mutations, to amplify both strands of a plasmid template. In addition to containing...template plasmid, these PCR products are directly transformed into bacteria expressing recombineering proteins...fine, especially using plasmids in the 10-12 kb range. REPLACR-mutagenesis also requires making your own...

...Gene X using shRNA expressed from a plasmid   Figure 1: Expression Level of Gene X The results shown above...Referring back to the experiment associated with Figure 1, the independent variable is the shRNA. Therefore ...the protein.   In analysis of the result in Figure 1, an internal control, such as the GFP-expressing Plasmid...control and is consistent with the result in Figure 1 could look like this: Figure 2: Expression of Plasmid...plasmid.  Let us now revisit our experiment. In Figure 1, it appeared as though the shRNA did not knock down...scientific inquiry and investigation. References: 1. The problem of pseudoreplication in neuroscientific...effects of the transfection itself (i.e., the vector used in transfection, transfection reagent, or transfection...

...family including human immunodeficiency virus type-1 (HIV-1), can infect both dividing and non-dividing cells...promoter, and the desired gene (transfer vector plasmid), 2) the HIV-1 genome containing all viral genes...have been devoted to develop efficient and safe HIV-1-derived lentiviral vectors due to their ability to...lentiviral vectors consisted of three plasmids encoding 1) the lentiviral vector genome which was composed of...generation because this vector requires only three HIV-1 genes (gag, pol, and rev) for production. Third generation...several protocols that have been described (protocol 1, protocol 2, protocol 3) to find the one that will...to additional protocols and plasmids.  References 1. Miller, A. DUSTY, and C. A. R. O. L. Buttimore. "...

... handed out at the event). Program 0 - 1:24 - Intro 1:24 - 29:31 - Reproducibility Overview - Jeffrey... 49:45 0 1:18:45 - Reagent Development - Steven C. Almo, Institute for Protein Innovation  1:18:45 - 2...reproducibility As Jeffrey Flier points out in his talk (1:24 - 29:31), there are a variety of ways of defining...enterprise towards greater reproducibility. Cause 1: Inability to repeat previously performed experiments...the Institute for Protein Innovation (IPI) (49:45 - 1:18:45), there are also initiatives to create more ...found. Consider the hypothetical situation in Figure 1 where many scientists are performing similar experiments...using the actual tool that you ordered and not a random aliquot pulled from a freezer. As you’ll see in...