...Determine ultrasensitive and absolute nucleotide concentration of starting material AAV Titrations Allelic...the primers can anneal to the single stranded DNA template strands. DNA polymerase is also stable at this...is a method that provides ultrasensitive and absolute nucleotide concentration, unlike qPCR where results...currently uses ddPCR for AAV titrating. You can learn all about ddPCR for AAV titration and find helpful tips...template DNA to amplify from and unique single stranded DNA primers that bind upstream (5’) and downstream... process as DNA polymerases require an existing strand of DNA to add nucleotides to.  Using these reagents...reaction:  Denaturation- To amplify DNA, the two strands of the template DNA first have to be separated....

..., expressing Ad E1A and E1B, can be transfected with your transfer plasmid, carrying your gene of interest... performed 12–18 hours post-transfection to help remove toxic transfection reagents and keep the cells...infectious titer, meaning the concentration of viral particles that can transduce cells, which can vary based...the AAV Transfer PlasmidAAV Variables That Matter AAV protocols on addgene.org  AAV Titration by qPCR ...TechnologyAAV Titration by ddPCRAAV Purification by Iodixanol Gradient UltracentrifugationAAV Production ...helper plasmids, commonly called “triple plasmid transfection” (Figure 1).    Figure 1: Overview of key steps...production, iodixanol gradient purification, and AAV titration by qPCR.   Pro tip! An optional media exchange...

...in an unmodified bacterial strain would result in read-through translation of many stop codons and detrimental...terminating translation in response to the presence of TAG sequences in ORFs. In this bacterial strain, TAG ...mutagenesis. In the C321.ΔA strain, this TGA will be recognized by Sep-tRNA and translated with a specific phosphoserine...one of the most inclusive databases of post-translational modifications, identifies a staggering ~250,... enabled researchers to create an orthogonal translation system (OTS) for the incorporation of phosphoserine...system defined three components required for translation (Figure 1): A tRNA that recognizes a specific...elongation factor that allows proper extension and translation of the pSer-incorporated polypeptide. These ...

... performed on whole-cell extracts from stable pools of A549 cells transduced with Cas9 expressing lentivirus...generated from A549 cells transduced with lentiCas9-blast. A549 cells were transduced (MOI = 37) and selected...generate DNA duplexes with one strand from the edited cells and one strand from the control cells, which...gene in the BRAF-gRNA transduced cells, and no such edits in the cells transduced by the control GFP-gRNA...generated A549 cells stably expressing Cas9 via transduction with Cas9 lentiviral vector, lentiCas9-Blast... Figure 1: Cas9 expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9 Western blotting...considering that A549 cells are relatively easy to transduce. I ended up with too many Cas9 integrations per...

...writers have been curious about the capability of transforming the genetic code of living organisms and its..., the father of scientific fiction, was also a trained biologist. This is apparent in his work The Island...that Streptococcus pneumoniae bacteria could be transformed from one organism to the other. This is now famed...explained how the four letters of the DNA get translated into the twenty amino acids. Once scientists ...what would happen if these corporations would extrapolate genetic engineering to exploit human beings. ...storyline involves the resurrection of dinosaurs by extracting the DNA from fossilized amber and supplementing... DNA sequences while FokI then induces a double strand break. These breaks can then be repaired by homologous...

...in special strains of E. coli that tolerate the expression of the toxin gene. One such strain is DB3.1,...popular lab strains, such as NEB Stable, JM109, XL1 Blue or XL10 Gold, that are F’. These strains could possibly...out our blog post on Common Lab E. coli strains for more strain info. Additional cloning resources As cloning...available for approaching it. These include the traditional restriction enzyme/ligase-mediated method, the.... One of the most time-consuming aspects of traditional cloning is the identification of clones that ...poison, locking up DNA gyrase with broken double stranded DNA and ultimately causing cell death. ccdA, another..., et al. showing the pKIL 18/19 vectors and demonstrating the concept of restriction enzyme-mediated disruption...

... The central nervous system (CNS) orchestrates complex processes enabling organisms to control their ...of common genetically encoded neuronal tracers Neuronal tracers such as epitope tags and fluorescent proteins...also been shown that dendrites in the stratum oriens and the stratum lacunosum moleculare were better resolved...molecules in your favorite neuronal population and track connections to that population. Limitations of ... used for protein localization, isolation, and tracking experiments (Rizzo, 2009). With many different...molecules or structures at once (Shaner, 2007). In contrast to epitope tags, FPs are bigger and their fusion...Spaghetti Monster - A new and improved neuronal tracer With the limitations of epitope tags and fluorescent...

...share stats and strategies with tech transfer offices. Drop us a line at techtransfer@addgene.org. Albert...Addgene Help Center: Tech Transfer Technology Transfer Information Tech Transfer News: A Reintroduction ...Addgene account for Tech Transfer Offices. They’re packed with tech transfer resources (summaries, electronic...question? Refer them to Addgene’s Tech Transfer Team: techtransfer@addgene.org Congratulations to the Golden...requests from Addgene MTA: material transfer agreement Technology Transfer: the exchange of intellectual property...addgene.org identifies the hold-up. Your Material Transfer Agreement is stuck in “pending.” Fear not, there... to make MTA turnaround faster with technology transfer offices (TTOs). The latest of these efforts is...

...students first transform the S. cerevisiae met deletion strains and then isolate transformed strains on media...overexpression plasmids. Students next transform deletion strains from the reference organisms with the...class research project, students transform S. cerevisiae met deletion strains with overexpression plasmids...of the plasmid-encoded MET gene allows the transformed strain to grow in the absence of methionine (i.e... successfully transformed with the plasmids, because the S. cerevisiae deletion strains are ∆ura3 mutants...MET/Met genes from the plasmids allows the transformed strains to grow in the absence of methionine. Since...undergraduate research questions. The idea is straightforward. Instructors first select a multi-gene pathway...

...their genome, some transgenic strains are difficult to breed. Some problems with transgenic mice that can ...expression level of the transgene. In existing strains of mice, the transgene can be maintained on a hemizygous... the transgene in order to see an effect. Hemizygous mice can be included in breeding strategies identical...different ways. Prior to engineering a transgenic strain, the promoter being used should be tested in cell lines...each mutated allele.  Cre-lox expressing strains Cre-lox strains are a common example of mice that are derived... mice are made from crossing a strain that expresses Cre with a strain that expresses a floxed gene of...are many types of genetically engineered mice: transgenic mice, knockin and knockout mice, and conditional...

...Förster Resonance Energy Transfer, FRET. Additional opportunities: Tracking intracellular pathogens In addition...and then transported in vesicles via the secretory pathway to their subcellular or extracellular destination... a part of an organelle or involved in intracellular transport pathways, it is possible to map the internal...can be gained by understanding the location and transport of the protein within a living cell. Analyzing...Analyzing differences in protein localization and transport between healthy and diseased states can also provide...a fluorescent protein fusion, it is possible to track your protein of interest from its production site...the end of their life cycle, proteins may be transported through the vesicles to the lysosome where they...

...which ensures the editor will only be transiently present in transduced cells, limiting off-target editing... dCas9 fused to a DNA methyltransferase — that can drive lasting transcriptional silencing (Nuñez et al...recruit and stimulate the endogenous methyltransferase. The strategy is generalizable to a variety of DNA-targeting... (cryo-EM) protocols typically require high concentration and purity of the target molecule, which can...developed a technique called Magnetic Isolation and Concentration (MagIC)-cryo-EM that combines enrichment and...detergents like CHAPSO, while requiring a lower concentration of sample — nearly ten times less protein. While...virus-like particles (eVLPs) offers an option to transduce cells directly with ribonucleoprotein cargo. eVLPs...

...blog posts: Extracurricular Activities for a Strong Science Career Path 7 Simple Strategies to Resolve ...Good Manager Anyway Developing Transferable Skills During Science Training ...key paper, or figuring out a career path? Many trainees are developing Individual Development Plans (IDP's...then formulate a plan to further develop your transferable skills and reach your career goals.      Although...used in isolation. Without clear dialogue between trainee and PI, it’s easy to miss out on beneficial opportunities...DePace holds yearly planning meetings with each trainee to create a supportive, growth-oriented lab culture... each of which is filled out separately by the trainee and PI before the meeting. These worksheets, summarized...

...CRISPR/Cas9 editing strategies in C. elegans As brilliantly and irreverently illustrated in the parable of...development of toolkits such as the SapTrap CRISPR/Cas Toolkit and SapTrap-SEC kit for C. elegans genome engineering...encode the gRNA transcript and the repair template of your choosing. Tools such as SapTrap Builder greatly...Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis...Emerging CRISPR/Cas9 editing technologies have transformed the palette of experiments possible in a wide...experimental manipulation and screening.  A co-selection strategy for CRISPR editing in C. elegans improves efficiency...Jordan Ward, UCSF. In 2015 (Ward, 2015), I demonstrated that selection for repair of a temperature-sensitive...

...Monitor neo-substrates Small molecule degraders may recruit CRBN to nonstandard or “neo-substrates.” We advise...al., Huang et al., Weintraub et al.), washout experiments (Nabet et al. and Weintraub et al.), chemical...et al.), transcriptional and epigenomic profiling studies (Nabet et al., Erb et al., Weintraub et al.),... et al., Huang et al., Weintraub et al.), knock-in (Nabet et al. and Weintraub et al.), and in vivo studies...degradation of endogenous proteins is an exciting strategy, it is difficult to apply this technique to oncoproteins...limitation, we created a generalizable tag-based strategy, which we refer to as the degradation tag (dTAG...targets in time scales that are not possible with traditional genetic approaches. In addition, the dTAG system...

...hiPSCs for clinical purposes: mRNA transfection- this allows transient expression of the reprogramming factors...cells, thus expressing additional EBNA-1 transiently. The transiently expressed EBNA-1 increases protein expression...is easily and routinely drawn from patients in contrast to the process of collecting fibroblasts, which...insertional mutagenesis, which is the integration of transgenes into the genome that can cause disruption of ...non-integrating RNA virus Episomal vectors- extrachromosomally maintained DNA plasmids that express reprogramming...reprogramming factors To date, the mRNA transfection method has not been used successfully to derive iPSCs...encoding reprogramming factors behave as separate extrachromosomal elements in the nucleus of the target cell...

...required for hok translation. Here, Mok and Hok translation is coupled. Translation from the Shine-Dalgarno...more stable than the methyltransferase. When a plasmid is lost, the methyltransferase is degraded and/or...Science translational medicine 7.289 (2015): 289ra84-289ra84. PubMed PMID: 26019220. PubMed Central PMCID... by binding the GyrA subunit of DNA gyrase and trapping it in the cleaved complex. This results in DNA...ccdB gene die off and a higher percentage of transformants contain the ccdB-less construct. Other toxin-antitoxin...introduce multiple genetic modifications in the same strain. MazF expression is under control of the AOX1 promoter...composed of restriction endonucleases and methyltransferases. Restriction endonucleases cleave DNA at ...

... cargo Optogenetic proximity labeling and TransitID Tracking cellular history with protein ticker tape...also incorporated LOV-Turbo into TransitID experiments.  Find TransitID plasmids here! These two developments...Dynamic mapping of proteome trafficking within and between living cells by TransitID. Cell, 186(15), 3307–3324...in vitro and in vivo. Incorporating reverse transcriptase (RT) variants, PE6a and PE6b are 500–800 bp ...37612276.   Optogenetic proximity labeling and TransitID By Mike Lacy Proximity labeling can identify protein...naturally present in experimental systems. They demonstrated LOV-Turbo in neurons, in pulse-chase experiments...plasmids here! Next, Qin and Cheah et al. described TransitID, where two proximity labeling enzymes, TurboID...

...of Pennsylvania Vector Core. Transfections are performed using the transfer plasmid, a plasmid encoding...are purified by iodixanol gradient ultracentrifugation and concentrated to purity and titers adequate for...ddPCR) titration protocol (modified from Wang et al., 2018). Briefly, 293T cells are transduced with serial...PCR with primers targeting the transfer plasmid used in the transfection. PCR products are analyzed by ...cell cultures are inspected for sterility. Transducibility Some viral vectors are tested in vitro and ...images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre (55634...cleared by low-speed centrifugation and then by filtration through a 0.45 µm polyethersulfone (PES) membrane...

...mammalian analogue of bacterial transformation is transfection. In stable transfection, DNA from the plasmid is...vectors delivered by transformation and transfection and left the business of transduction to our Viral Vectors... Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection Plasmids 101: Choosing...to retain the vector as cells divide. In transient transfection, the vector remains episomal, meaning it...vector replication at all. In these cases, transient transfection may be sufficient for your needs even without...called transfer vectors. The gene of interest is cloned into a shuttle vector for transfection into producer...has seen before, showing how DNA sequences are translated into amino acids. And I was astonished that the...