...way to begin the search is by using selection strategies, where only cells that have gained or lost a ...plasmid cloning.  But oftentimes, a selection strategy is not enough on its own to help you find your...plasmid. In many cases, you will need a screening strategy as well. In a screen, you aren’t killing off a..., then screen Why add a screen to your cloning strategy? A screen will help you more easily identify successful...information (more on this below). Plating the transformation on ampicillin plates containing X-gal allows...Fortunately, for the lab researcher, when the substrate X-gal is broken down by 𝛽-galactosidase, it turns...blue-white screening at Addgene! Some bacterial strains contain lacZ in their genome, and scientists have...

...repeats (ITRs) are what makes an AAV transfer plasmid an AAV transfer plasmid. ITR sequences are 145 bases...read through this challenging sequence. Transgene The transgene is the gene that will be delivered by the...Most AAV transfer plasmids have a promoter, but this element isn’t required when the transfer plasmid ...control transgene expression. Lox sites are directional 34 bp sequences that can flank the transgene and ...(WHP) Posttranscriptional Regulatory Element (WPRE) forms a tertiary structure when transcribed which ... nuclear export of RNA and RNA translation, and promotes RNA transcript longevity. The polyA is located...proteins needed for the virus to replicate,  and the transfer plasmid which contains the viral genome. In today...

...imaginations to use in this world where we are often trained to follow protocols, guidelines, and standards.... an odd or quirky lab story you’d like to see portrayed in LEGO form, feel free to share with us at BrickingScience...

...contain plasmids deposited at Addgene from the abstract page. Check out that beautiful Addgene “Find Plasmids... Plasmids” badge in the image below. If an abstract on PubMed has the “Find Plasmids” badge, you’ll know... your PubMed account and are browsing article abstracts you should see the “Find Plasmids” box on papers...

... don’t wait. Start communicating now. These 9 strategies will get you started:  Subscribe to Career Blog...internships to cover their annual meetings. 8) Training – Get a formal education Some people get into ...journalism graduate schools where you may take a science track. If you don’t want to commit time and money to a...engagement for institutions and the Royal Society gives training in media skills, writing about your research and...learning courses at the National Council for the Training of Journalists (NCTJ) or the Alan Alda Center ...

...different strains of E. coli that can be used for transformation. The preferred E. coli strains for storage...packages have spent days to weeks in transit and some have been transported across oceans and continents. Each... The transformation After the DNA has been received and stored, it is put into E. coli strains that have... treated to easily take in plasmids (transformation). Transformed bacteria are then grown up on antibiotics...received is immediately transformed and stored. Some samples need to be transformed with a micropulser using...samples require a reduced antibiotic concentration or need to be transformed into different cells than were ... performs an average of 140 transformations. The lab typically transforms newly deposited samples from...

...body, like the gastrointestinal tract. Require Administration of Substrate: Some luciferase reporters, such...therefore produce signals in ultrasound imaging. Ultrasound is an attractive imaging platform for in vivo...coli aided in ultrasound imaging of the mouse gastrointestinal tract. The E. coli strain Nissle 197 (ECN...NanoLuc, require injection of substrate to produce bioluminescence. Substrate pharmacokinetics and biodistribution...because  ultrasound devices are inexpensive, widely available, and have deep tissue penetration and high...are invisible to ultrasound, which allows for differentiation of arg1 and arg2 ultrasound signals. This ...because it’s a probiotic strain that can colonize the mammalian intestinal tract and has been used for 100...

...type VI-D enzymes are the latest tools in the transcriptome engineering toolbox. How were Cas13d enzymes...small hairpin RNA interference, dCas9-mediated transcriptional inhibition (CRISPRi), and Cas13a/Cas13b RNA...only time will tell how large the genome and transcriptome engineering toolbox will be. It is, however,...community. References Konermann, Silvana, et al. “Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR...Editing with REPAIR Get tips for using Cas9 Transcriptional Activators Learn about Cas13a (C2c2) Characterization...

...positive perspective on the transition to a post-academic career. This training was “included” with their...doing an inadequate job of supporting science trainee transition into diverse, successful careers.  We’re ...The pandemic has demonstrated that, more than ever, we need all of our science trainees to be successful...are intended to be widely disseminated by “train the trainer” programs.  Please join us for the pd|hub ... continue attracting and launching science talent we must ensure that career skills training is a required...all science training programs. I believe in the academic apprenticeship system as a training ground for...even doing a very good job of providing skills training for scientists destined to continue in academia...

...serotype. Additionally, we check viral transgene expression in transduced cells whenever possible. This post... for transgene expression. Once packaged in AAV, this pre-recombined DNA could lead to transgene expression... users titrate their AAV vectors to find the optimal dose that allows for sufficient transgene expression...to make sure they are present at a very low concentration. So how do we make sure to catch all of these...particles (Figure 1). Briefly, packaged DNA is extracted from purified, DNase-treated AAV and submitted...with AAV-68544 to validate our method. We then extracted the DNA and processed the mixed sample in our ... research-grade materials. Addgene is going the extra mile and pioneering the systematic use of this new...

... Michael Davidson and Roger Tsien Commemorative Travel Awards. These awards are intended to help scientists...research with the academic community by funding their travel to a conference of interest. In this post, we’re... Michael Davidson and Roger Tsien Commemorative Travel Awardees: Xin Zhou and Fatima Enam. There were ...protein known to oligomerize when exposed to ultraviolet light. To do so, she generated fusion proteins...of an enzyme’s active site. In these fusions, ultraviolet light exposure caused oligomerization of the ...still block enzyme function upon exposure to ultraviolet light. Previous iterations of FLiPs only used...Cdc42, Raf1, Mek1, Mek2, Cdk5, and Cas9. Xin has demonstrated the utility of these FLiPs in a variety of research...

...vivo.  But what ultrasound lacked was reporter genes that could produce ultrasound contrast. That is, until...before imaging with ultrasound to observe gas vesicle-specific ultrasound contrast where the bacteria ...via regular intraperitoneal doxycycline injections to enable their tracking with ultrasound imaging (Fig...the transient cotransfection of the mARG plamsids into HEK293T cells using a chemical transfection reagent... scatter sound waves and thereby produce ultrasound contrast. We (the authors) are currently working on...also straightforward: after the E. coli have colonized tumors, inject L-arabinose intraperitoneally and...phase contrast microscopy, GVs visible in cells via TEM, and purified GVs under TEM. (c) Ultrasound imaging...

...phosphatase-accessible. Kinase Translocation Reporters (KTRs) Kinase Translocation Reporters (KTRs) are genetically...particularly important for regulating signal transduction and measuring kinase activity at the single-...provides an alternative tool with their Kinase Translocation Reporters (KTRs) whose cellular localization...of two fluorescent proteins with very specific spectral properties that limit which fluorophores can be... A KTR is comprised of a fluorescently-tagged substrate for a kinase of interest which is further fused...activity is enhanced, leading to cytoplasmic translocation of the reporter. As a result, the KTR is shuttled... simple when fluorescent proteins with limited spectral overlap are used together. Second, KTRs are easy...

...solve the issue of “tiny vector, big transgene” by just straight up packaging an oversized genome of ~... 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...overviews of three split vector strategies plus one additional split vector strategy for increasing the size ...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...

... recent works, transdifferentiation is usually carried out by expressing transcription factors specific...successfully transdifferentiated into neurons and β-cells [40-42]. Reprogramming and transdifferentiation: When...18818365. PubMed Central PMCID: PMC3987909. 6. Ban, H., et al., Efficient generation of transgene-free human...Cells. Stem Cells Transl Med, 2016. 5(5): p. 561-71. PubMed PMID: 27013738. PubMed Central PMCID: PMC4835250...PubMed Central PMCID: PMC4363833. 35. Eguizabal, C., et al., Dedifferentiation, transdifferentiation, and...PMID: 20364121. PubMed Central PMCID: PMC2877635. 39. Davis, R.L., H. Weintraub, and A.B. Lassar, Expression...redifferentiated to a different cell type. Transdifferentiation In this process, differentiated adult somatic...

...expressing a novel reverse transcriptase (RT), MarathonRT. Reverse transcriptases are critical tools for ...!  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...29109157. Nanobody toolkit to monitor retrograde transport at the cell surface Article contributed by Marcy...antibodies. These properties make nanobodies easier to transform and express in bacteria, which, in turn, makes...

...restriction digest of plasmid DNA, and bacterial transformation. And more plasmid-related educational resources...reagent storage and inventory, managing orders, tracking students and research projects, electronic lab...share protcols, sign-up for shared equipment, and track inventory and orders. Quartzy caters to life science...features, and reference tables (i.e. antibiotic concentrations, commonly used primers). We also encourage ...

... cross-neutralization occurs in the P.1 and B.1.351 variants tested. Image from Garcia-Beltran et al.,...high-throughput SARS-CoV-2 pseudovirus neutralization assay. Neutralization was quantified in terms of luminescence...Image from Garcia-Beltran et al., 2021 with permission.   Difference in neutralization after one dose...compare so many different strains. It was also the first to perform neutralization studies after just the...MIT, and Harvard, and his lab found that the neutralization effectiveness of current vaccines differed ...differed depending on the variants (Garcia-Beltran et al., 2021). Antibodies from individuals who received both... doses of the Moderna or Pfizer vaccine could neutralize many variants, including the B.1.1.7 (Alpha variant...

...surprising because in vivo transduction of AAV serotypes generally tracks between strains of mice. This suggests...PHP.B has a >40-fold increase in CNS transduction and transduces the majority of astrocytes and neurons... mice whose CNS was transduced by PHP.B but absent in mice who were not transduced by PHP.B. The Deverman...coding regions. In total, they tested 13 strains, with seven strains identified as permissive and six as non-permissive...enhanced PHP.B transduction rates. The group also determined the effect of mice strain specific Ly6a SNPs...permissiveness While PHP.B has impressive CNS transduction in some strains of mice, these results are not reproduced...in different mouse strains. The in vivo selection for PHP.B in C57BL/6J mouse strains likely contributed...