...before you can start transducing your virus. Let’s start there!     Figure 1: Flowchart showing...optimized your transduction conditions, you’re ready to transduce! Addgene has a transduction protocol you... the pooled library is transformed into E. coli for amplification. Transformed E. coli are allowed to ...functional titering and optimize transduction efficiency. Transduction efficiency can vary with titer, ...cells to transduce, you may want to consider spinfection. Spinfection is the process of transducing cells...this post, I’ll walk you through how to prep and transduce your pooled library.  Pooled libraries Scientists...sequencing to confirm library representation. Transfection of the DNA pooled library and appropriate packaging...

...mammalian expression from human elongation factor 1 alpha Constitutive  Tends to give consistent expression...tetracycline operators. Transcription can be turned on or off depending on what tet transactivator is used. UAS...for in vitro transcription, the promoter drives either the sense OR antisense transcript depending on ...This element is responsible for initiating the transcription of your insert into RNA. In practice, the term...controls the binding of the RNA polymerase and transcription factors, therefore promoters play a large role...

...necessary.   Tips for barcoding and tracking samples 1. Start with the tube - Barcoded tubes come in many...packages have spent days to weeks in transit and some have been transported across oceans and continents. Each... 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... performs an average of 140 transformations. The lab typically transforms newly deposited samples from...delivered by a mail courier and then the journey of transformation and storage begins. Some samples are submitted...long-term storage in our facilities, every sample is transferred to a 2D barcoded tube (see next section) and ...

... such a popular model organism? Zebrafish (Figure 1) were initially recognized as a useful vertebrate ...strategy uses a transposon called Tol2 to randomly integrate foreign DNA into the genome. Non-random integration...et al., 2019). Generating zebrafish transgenics Creating transgenic organisms by introducing foreign DNA...Danio rerio) have been used since the 1930’s in a range of biological studies, including investigations ... to produce abundant, externally fertilized, transparent progeny (Figure 2). These features give it a ...individual to maintain. Also in contrast to mice, the transparent zebrafish embryo develops outside of the mother...sequenced genome also permits “whole-genome/transcriptome” studies, in which, for example, the activity...

...impacts of neural circuits on behavior. Figure 1: An overview of the chemogenetics system. Why use...approved drugs so that your results are viable for translation to human patients. Picking receptors The receptor... specifically designed Gi-coupled receptor in transgenic mice. Nature Biotechnology 17:165–169 . https...

...inclusion bodies and is folding properly. References 1. Angov, Evelina, et al. "Heterologous protein expression...structures in RNA transcripts Effects on transcription (remember, it’s not all about translation - e.g. codon... you cannot choose among the possible codons at random and expect your sequence to express well in any...therefore may need fewer resources for the entire translation process. For example, during high growth rate... tRNAs. For example Tuller et al. found that translation efficiency is well correlated with codon bias...tRNAs, different regions of the protein may be translated at different rates. The ribosome will move quickly...the ribosome stalls, this may give the swiftly translated regions a chance to fold properly. For example...

... education and scientific writing.     References 1. Martz, Colin A., et al. "Systematic identification...’s group at the University of California, San Francisco, is approaching the Ras problem by exploiting ...lines. Further, for each plasmid in the kit, the transcript most commonly found across all TCGA tumor samples...

...plasmids are passed down through generations (Figure 1). These so called “plasmid addiction systems” require...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...systems have been used as biological tools for a wide range of applications in molecular biology or in vivo ...ccdB gene die off and a higher percentage of transformants contain the ccdB-less construct. Other toxin-antitoxin...composed of restriction endonucleases and methyltransferases. Restriction endonucleases cleave DNA at ...specific nucleotide sequences. To counter this, methyltransferases add methyl groups to bases at specific nucleotide...

...such as model systems and primary cells. References 1. Doench, John G., et al. "Rational design of highly...prevent efficient transcription from sgRNA targets. CRISPRi is effective in a narrow range around the transcription...two sets of sgRNAs; A, the top 3 ranked sgRNAs, and B, the next 3 ranked sgRNAs. Dolcetto was able to discriminate...Cas9 derivatives. Unmodified Cas9 causes double stranded DNA breaks that commonly result in a complete ... lab selected sgRNAs in this optimal window and ranked them based on their optimized sgRNA rules and potential...gene expression, dCas9 can also be used for transcriptional activation. Activation of specific genes allows... strategies have been developed to recruit transcriptional machinery to dCas9 and its RNA directed binding...

...determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV ...of Pennsylvania Vector Core. Transfections are performed using the transfer plasmid, a plasmid encoding...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...filtration through a 0.45 µm polyethersulfone (PES) membrane. The preparations are then aliquoted, frozen, ...from Wang et al., 2018). Briefly, 293T cells are transduced with serial dilutions of a lentiviral vector,...

...determined for your sample. Typical ratios may range from 1:1 to 1:6. Add the diluted PEI-MAX to the diluted...solution at -20 °C. 1 mg/mL PEI-MAX Add 1 g of PEI-MAX powder to 900 mL deionized water in a 1 L bottle and ...to mix. Add 450 µL of 1 mg/mL PEI-MAX to the second tube of 6 mL BCD TFX (for a 1 mg/mL stock solution ...antibody At 168 hours (1 week) post-transfection, harvest the antibody. Transfer the HEK293 cells and media...February 18, 2022 Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells Day 7: Harvest...at -20 °C. Procedure Section 1: Seeding cells The day prior to transfecting, seed a 108 mL culture of HEK293.... Cap the tubes and incubate for 1 h in the 37 °C bead bath. Transfer the PEI-MAX and recombinant antibody...

...antibiotic selection. Essentially, this gene is a 2-in-1 providing both bacterial and eukaryotic selection,...in a plasmid. During the propagation and/or transformation process, some bacteria will either never receive... is nifty because the recovery period after transformation (the dreaded 60 minutes shaking at 37 degrees... available, cost-effective, timesaving for transformations. Cons: Prone to satellite colony formation ..., interchangeable with Amp, timesaving for transformations. Con: Expensive.   Kanamycin Kanamycin, or...selection gene. However, it does require a slower transformation recovery period than amp or carb (60 minutes...confers mammalian resistance to G418. Con: Slow transformation   Spectinomycin/streptomycin Spectinomycin...

... of low-toxicity RNA editing tools.   Fig. 1 Schematic of luciferase RNA knockdown assays using...PRIME, PASTE relies on single-stranded nicks instead of the double-stranded breaks used in other insertion...target. In addition, when they looked at the transformed E. coli expressing Cas7-11, they found no evidence...broadly similar to shRNA and Cas13 systems in a range of mammalian cell lines. However, in those same ... of landing sites via Cas9-directed reverse transcriptases, followed bylanding site recognition and integration...

... done and we’re onto the next story. Figure 1: Addgene’s Agile Approach Sharing is caring There...to be translated into Modern Greek to be easily accessible today. Roughly speaking, we translated our software...see these improvements, you are benefited with reassurance your plasmids will not get lost in a freezer ...Another advantage of Scrum is that it provides transparency to everyone involved about the status of each...accelerating research and discovery. We work with transparency, gladly share our knowledge, and love to collaborate...

...processed since the declaration, only a small fraction (<1%) of authors have asked for advice about the scope...supporting datasets must be logically annotated and arranged, as in an actual publication and not require interaction...responsible for storage.  If datasets are too large to transfer to a journal repository, they are likely too large...albeit unintentional, tendency to work against transparency. This opens science to charges of elitism and...opportunities for research as well as increasing transparency. Thank you to our guest blogger! Jim Woodgett...Will Pay for Public Access to Research Data? - by Francine Berman and Vint Cerf (Science, August 2013) PLOS...

...fun together! Laura’s Top 10 Event Planning Tips! 1. Take suggestions Some of Addgene’s best events have... elaborately pranking each other, dressing in costume, and holding competitions (ranging from cook-offs...orderly and timely fashion. 9. Ask for help My grandma used the old adage “many hands make light work”..., evite, posters, megaphone, or your company’s intranet. Don’t forget about the power of simple word of...

...antibody bonding strengths.    . Steps of an IP: 1) Protein extracts from cells or tissues contain a ... protein of interest, such as a sample transiently transfected or stably infected with the protein of ...downregulated, examine a protein’s stability or post-translational modifications, or study how a target protein...experiments using a tagged protein may not be translatable to the endogenous system and will need to be...

...References 1. Haery, Leila, Ryan C. Thompson, and Thomas D. Gilmore. "Histone acetyltransferases and histone...popular papers, but looked for papers from a wide range of authors. Just start writing When I first started...drafts of the manuscript. This is the time to transform the document into something cohesive-- change ...Tips for Improving Your Next Manuscript 6 Tips for Grant Writing Resources on Addgene.org Check out Our...

... reached labs in over 75 countries. Figure 1: SARS-CoV-2 proteins and other components. Image from...loop-mediated isothermal amplification after reverse transcription (RT-LAMP) and a colorimetric readout that can... 45 minutes. The team deposited the reverse transcriptase, protease, and Bst polymerase constructs from...toolkit include ones for expressing MMLV reverse transcriptase and Taq polymerase that can be used in a one-step...

...two (Knutson et al., 2010a,b).      Fig. 1: Undergraduates in Provost’s CURE prepare to analyze...). But is it possible to bring the kinds of transformative educational experiences afforded to those fortunate... now connects MDH-focused CUREs at 50 schools, ranging from R1 institutions to community colleges, and...NADH production.   CUREs are beginning to transform undergraduate education in the sciences by making...