...curves from our recent amplification of the Brunello library are also shown in Figure 3. I hope you’ll agree...would be a good time to talk about the amplification and verification processes we use to ensure high-quality...your own experiments. Library amplification 101 Library amplification seems straightforward: E. coli ...transformed with the library DNA, they grow and replicate, and then their DNA is harvested, much like one...library has its own depositor transformation and amplification protocol, but the overall workflow is similar...ll need to remember when undertaking library amplification and sequencing. Maintain representation of the...electrocompetent Stbl4 cells when performing amplifications, and we use electroporation to ensure a good...

...Lebbink RJ, Rehmann H, Geijsen N. Cell. 2015 Apr 23;161(3):674-690. doi:10.1016/j.cell.2015.03.028. PubMed. ...protein-only transduction method and its potential applications to CRISPR/Cas9 genome editing.   Protein-only...the HIV protein Tat. Although CPP fusions can translocate a cell membrane, the presence of the CPP may ...that components in the buffer used for protein purification must be helping the untagged protein enter cells...Diego D'Astolfo. CRISPR/Cas9: a potential application of iTOP Having tested iTOP using Cre-lox recombination...showed up to 70% resistance to diphtheria toxin, indicating a 70% frequency of biallelic mutations. Importantly...Importantly, hESCs retained their stemness, indicating that iTOP may be valuable for CRISPR/Cas9 stem cell...

...need to move around every 3-6 years—a short and exciting ride Complicated matrix structures are common...scientific and technical training is that these educational paths prepare you for a vast selection of career...tech transfer, management consulting, science communication, policy and the diverse options in nonprofit...science is much like industry but the “product” is publications—the pressure to produce is the same You get ...and teach students Collaborations may be less “complicated” - for example, easy to initiate a collaboration...is different about non-academia? Scientific publications are a nice to have, not a need to have Must ...

...stressed or aging cell populations. Figure 3: Micrographs of wild type (top panels) and cells with...infection, because this setup allows for experimental replicates for each of the gRNAs. The idea was to assign...cassette is removed, the effector is expressed. By truncating one of the recombination sites in the construct...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ... reporter (blue) and stained with PI (pink) to indicate cell death. Image from Diessl et al., 2020. ...https://dx.doi.org/10.15698%2Fmic2020.04.713 Truncated TALE-fluorescent protein fusions as a DNA staining...protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry. In comparison...

... for a Successful Collaboration. PLoS Comput Biol 3:e44 . https://doi.org/10.1371/journal.pcbi.0030044...Collaboration and creativity are pivotal in writing publications/grants too. As described by several works (Wuchty...backgrounds.” This work outlines the need for clear communication, fair credit through authorship, and acceptance...remotely. The authors stress the fact that “miscommunication, a lack of leadership, and inappropriate tools...approaches can lead to frustration, delay of publication, or even the termination of a project.” Take ...setting. Check out the blog post “Fostering Communication in Remote Collaborations” to get more ideas....Lewis ASL, Weyhenmeyer GA, O’Reilly CM, Lofton ME, Catalán N (2018) Ten simple rules for collaboratively writing...

...ready for the live audiences in your future.     3. Start something In almost every career talk I present...be a time when you need to speak publicly to communicate your work. One of my 10 tips for giving a great...; Graduate Student Funding Opportunities Open Education Database: 100 Places to Find Funding For Your ...

...Brandon Sanderson "The Mistborn series is the first 3 books of a larger epic of fantasy novels. Set in a...sometimes we don't."     - Jennifer Tsang, Science Communications and Marketing Coordinator Non-science favorites...times."     - Aliyah Weinstein, Marketing and Communications Manager Addgene's book club Indeed, reading...

...thousands of loops. These loops not only provide a 3-D structure for packing genomic DNA into nuclei, but...plasmids at Addgene Opperman J et al., Nature Communications, 2019. PubMed PMID: 31346176. Using LADL to...light-activated-dynamic-looping (LADL) system. The group fused dCas9 to a truncated CIB1 protein from Arabidopsis (CIBN), which they...

...Halorhodopsin plasmids . Archaerhodopsins Archaerhodopsin-3 (Arch) from Halorubrum sodomense is also commonly ...with single-spike temporal resolution. PLoS One. 2(3):e299. PMID 17375185 Kim B, Lin MZ. 2013. Optobiology...excitation of distinct neural populations. Nat Methods. 11(3):338-46. PMID 24509633 Lin, J. 2011. A User’s Guide...control of neural activity. Cold Spring Harb Protoc. 3:top102. PMID 21363959 Yizhar O, Fenno LE, Prigge M...derived from Volvox carteri. Nat Neurosci. 11(6):631-3. PMID 18432196...Channelrhodopsins: cation channels from Chlamydomonas reinhardtii ChR2 Widely used light-gated cation channel from...light of the correct wavelength, the pore opens, cations flow into the cell (yellow dots), and the neuron...

.../ I203R) (2) and PA-mRFP1(S146H / I161V / I197H) (3) were derived from DsRed and mRFP, respectively. When...PubMed PMID:19169259. PubMed Central PMCID:PMC2901231. 3.Verkhusha, Vladislav V., and Alexander Sorkin. "Conversion...up. Applications of photoactivatable fluorescent proteins Some of the most important applications for ...conventional FP imaging. PA-FPs are divided into 2 main categories - those with irreversible and reversible photoactivation...photoactivation - and have enabled sophisticated imaging techniques.     Irreversible photoactivation Irreversible...level of background fluorescence. For any PA-FP application, the brighter the fluorescence the better. More...photoactivation. A high contrast ratio usually indicates that a PA-FP has a low level of spontaneous fluorescence...

...and New Faculty" downloadable book. Skip to Chapter 3 for tips on how to develop your role as a lab leader...environment for research is totally dependent on communication amongst the team so getting everyone talking...

...in Vector Transgene Expression. Hum Gene Ther, 31(3–4), 151–162. https://doi.org/10.1089/hum.2019.274.... ITRs serve as the origin of replication and are required for replication, packaging, and vector persistence...viral rep (replication) and cap (capsid) proteins, which bind to the ITRs and initiate replication. ITRs are...structures, ITRs are prone to replication errors and disruptive mutations and truncations that can make vector ...binding site for Rep78 and Rep68 to initiate replication and a terminal resolution site that serves as...are noted. Created with biorender.com. ITR modifications and types  Since ITRs are one of the most important...important elements of AAV and rAAV vectors, modifications can drastically affect gene transfer efficiency...

...the filters on the right to find single, dual, and 3+ cutters. You can also sort enzymes by clicking on...function. You can also click one location in the plasmid and then a second location to select the sequence between...two separate locations in the linear sequence selects the sequence between the two locations and sends it...table includes feature names, locations, sizes, colors used to indicate them on the map, direction (if...promoters, origins, tags, and certain ORFs. Their sophisticated detection algorithm easily and precisely identifies...restriction enzyme annotations throughout the map at locations better aligned to their precise positions within...between the two locations and send it to the clipboard for copy and paste functionality. Sequence ...

...any DNA they were bound to (2) fragmented the DNA (3) used antibodies to specifically select for proteins...next-gen sequencing. ChIP is an incredibly useful application for exploring these interactions, and can give... protein interactions, including epigenetic modifications. But it's not an approach for the faint of heart...background information. Your options for this step are sonication, which uses high-frequency sound waves to break...break up the DNA, or enzymatic digestion. Sonication provides a truly random digestion, but is harsher ...to the selective nature of the cutting. While sonication is usually the preferred method, enzymatic digestion...concentration.  Protein Capture After crosslinking and sonication or enzymatic digestion, you’re ready to incubate...

...of the selection reagent). Cells should die within 3-5 days and resistant colonies appear in about 10-14... a bacterial origin of replication (ORI) will not allow for plasmid replication in mammalian cells, for... their bacterial counterparts, including how replication occurs and whether selection is necessary for...the nuclear genome, in the absence of plasmid replication, the foreign DNA will be degraded or diluted ...always mission critical to make sure the plasmid replicates in the cell, as many of these experiments are...expressed in trans within the cell for effective replication. Cell lines expressing the Epstein–Barr virus...antigen (293E or 293T cells), allow for episomal amplification of plasmids containing the viral EBV or SV40...

...great tool for someone starting cell culture work. 3. NEB Tools by New England BioLabs In my lab we do ...Gibco organizes its app into four categories: tools, analytics, education and data manager. The tools are...are five FREE science lab apps that have been educational and great for lab life. These apps prevent you...lab notebook. Lab Archives is a multipurpose application that allows you to share your data with colleagues...other nice features. This app also has a great educational section. This section holds the Gibco Cell culture...directions including which buffers to use and that indicates if a sequential digest is needed. The enzyme finder... to this app; the user can search by sequence, category of enzyme, or name to discover all the information...

... produce and distribute virus. Addgene is a 501(c)3 nonprofit and our plasmid service was started based...Addgene started distributing a small but growing catalog of ready-made AAV and Lentiviral preps. This new...directions. I would like to thank them for their time, dedication, advice and encouragement. Why virus? Why did...performs extensive quality control, generates  educational materials, and provides viral vector technical...answering questions on virus and helping provide educational resources on this collection. With their expertise... resource and providing fantastically useful educational content on viral vectors.  How Addgene’s Viral...about the new initiative and serving all the new educational content via the Addgene Blog and our expanding...

... lab E. coli K12 strains such as DH5alpha contain 3 methylases that recognize and methylate different ... one half of naturally occurring restriction modification systems that prokaryotes use to protect themselves... has genes encoding one or a few restriction modification systems containing methyltransferases that add... phage for instance). Aside from restriction modification systems, DNA methylation also plays an integral...integral role in regulating genome replication, repairing mismatched basepairs or small indels that occur...methylases) are independent from the restriction modification systems, yet can still affect whether certain...specifically associated with any restriction modification systems, their sequences may overlap with restriction...

... minutes of my hands on time and a total of about 3 hours. It was robust, capable of generating a wide...processing, these small DNA strands can form concatemers in later steps. This new NEB kit includes a step...fragments the DNA and prevents it from forming concatemers later. The "FS" stands for Fragmentation System...repair Adapter Ligation Clean up/Size selection Amplification Clean up  I found this kit easy to use because...DNA we started the process with, we got great amplification of our sample libraries. NEB also gave us a ...mostly used to make new libraries as opposed to replicating them like we do at Addgene), going to the workshop...

...engineered a DAP array for prime editing, achieving up to 3-loci MPE with similar high efficiencies, which is ...packaged into viral vectors for potential in vivo applications or multiplex CRISPR screening.   Using tRNAs...multiplex genome engineering of crops or allow for sophisticated CRISPR screenings that would perturb the unprecedented...and efficient in plasmid and viral vectors. In applications when repetitive tRNA sequences must be avoided...construct DAP array for multiplex gene editing applications, we provide detailed manual and online design...