We narrowed to 735 results for: RAN-1

...(RBD) of the viral spike protein.   Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and sequencing... lab has created a novel nanocompartment for transporting proteins in yeast by using murine polyomavirus...2A-Synaptophysin-mRuby from Liqun Luo’s lab expresses membrane tethered GFP to enhance axonal labeling and synaptophysin-tagged...expressing GCaMP6f with this promoter. Image calcium transients in PV interneurons with AAV-S5E2-GCaMP6f are ...

...understand these strange results by digging deeply into their data.     Fig. 1: Genomic fraction...Samples are divided into those of African ancestry(orange) and those of other ancestry groups (grey). ...): 250-252. Carlson J, Henn BM, Al-Hindi DR, Ramachandran S. Counter the weaponization of genetics research...

...experiments, to false conclusions.  Figure 1. Potential points where DNA errors can be introduced...accuracy across all of our plasmids and provide transparency in sequence discrepancies. While Addgene’s QC...

...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...

...designed to shuttle between species. Figure 1: Shuttle vectors are vectors that can function in ...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...

...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...

...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...

...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...

... 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,...

...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...

...sample’s absorption at 260 nm (A260). Figure 1: Characteristic absorption spectra for pure DNA and... that this simple procedure is often taken for granted. In this post, we take a moment to review what ...a characteristic absorption spectra across this range of wavelengths with maximal absorption at 260 nm...present in the sample also absorb light in this range of wavelengths (Figure 1B-D). These other molecules....  In common practice, a pure sample of double-stranded DNA should give an A260/280 ratio of ~1.8 and ...Proteins absorb light to varying degrees across the UV range used in NanoDrop systems. Peak absorption occurs...

... 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...

... 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...

...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...

...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...

...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...

... 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...

...commandments for making it all work – most of the time. #1 Thou shalt find enjoyment in your work If you are ...responsibilities. Another tip: don’t miss your grandfather’s 100th birthday, as your mom will never forgive...when you can’t get home, babysitting or running errands. Give help when you have time, and in return you...decent meal, fold their laundry, take public transportation, call a cab and remember their own school assignments...

...to an antigen. AdEasier-1 cells BJ5183 E. coli cells containing the pAdEasy-1 packaging plasmid. Adenovirus...enzyme PmeI . This construct is then transformed into AdEasier-1 cells (Addgene #16399) , which are BJ5183...plasmid pAdEasy-1 (Addgene #16400) is suitable for most purposes. For especially long transgenes, the use of...gene therapy landscape . Signal Transduction and Targeted Therapy, 6 (1), 53. https://doi.org/10.1038/s41392...icosahedral capsids and build new virions. Figure 1: Wild-type adenovirus and its genome. Genes over or...production system. Figure is based on the use of pAdEasy-1. Created with BioRender.com. AdMax™ AdMax™ is another...as a Zika virus vaccine . Scientific Reports, 10 (1), 3613. https://doi.org/10.1038/s41598-020-60238-5...

...for your first journal club to begin! Figure 1: Discussing next-generation sequencing at the Addgene...of current new and exciting literature. As I transitioned out of academia and away from the bench I certainly...

...can make this fun activity relate to STEM.” Figure 1 shows the activities we had for my school’s Math &...olds in a single evening. Grades K-5 cover a broad range of attention spans, skill levels, and STEM exposure...aspects of talking to 8th graders was the broad range of questions I received. I gave my presentation ...

...blood test for HMGB1, the high mobility group box 1 protein. Normally localized to the nucleus, if the...of diagnosis) is about 35 years, but it usually ranges from 20-50 years and sometimes longer. There is...be Used to Study Cancer Drug Resistance Plasmid Grant to Aid Cervical Cancer Screening in Ghana Additional...

...testing. You can find Nick on LinkedIn. References 1. Skloot, Rebecca, and Bahni Turpin. The immortal life...cell line contamination: Epithelial Mesenchymal Transition Is Required for Acquisition of Anoikis Resistance... to carry our independent quality control for a range of testing, from animal doping to forensics, DNA...

...Addgene's Chemogenetics Plasmid Collection . Figure 1: General overview of a chemogenetics experiment. Early...bidirectional control of neuronal activity. Table 1: Descriptions of DREADDs and their activity in neurons... while PSAMs and LMOs have an effect for only 0.5–1 hours after delivery. Chemogenetic Ligand The chemogenetic...Causal Cellular and Neuronal Biology. Physiol Rev, 98 (1), 391–418. https://doi.org/10.1152/physrev.00009.2017...Neurophotonics 11 (2), 021005. https://doi.org/10.1117/1.NPh.11.2.021005 PMID: 38450294 Coward, P., Wada, H... Gi-coupled receptor. Proc Natl Acad Sci USA, 95 (1), 352–357. https://doi.org/10.1073/pnas.95.1.352 PMID... 451–460. https://doi.org/10.1007/s00213-004-2017-1 PMID: 15765260 Farrell, M. S., Pei, Y., Wan, Y., Yadav...

... typically ranges from 8–10 kb, encoded on RNA (Figure 1). This RNA is reverse transcribed into the provirus...— contains transgene and LTRs (chimeric 5’ LTR) Packaging plasmid 1 — contains gag and pol Packaging plasmid...pre-infusion CAR T-cell products. Journal of Translational Medicine , 20 (1). https://doi.org/10.1186/s12967-022...been removed or mutated for increased safety. Figure 1: Wild-type lentivirus genome. Created with BioRender.com...genes (for example, vif , vpr , vpu , and nef for HIV-1). The LTRs flank all of these genes, and anything ...and third-generation systems, please refer to Table 1. Figure 4: Third-generation lentiviral plasmids. Created...heterologous enhancer/promoter such as CMV or RSV Table 1. Summary of key differences between second- and third-generation...

...complex. So how exactly does this work? Figure 1: APOBEC- and Cas9-mediated editing (ACE) reporter ... affecting protein translation of different reporter proteins. Protein translation requires the start ...compared to CRISPR/Cas9 edits that rely on double strand DNA breaks. Due to this inefficiency it is crucial...constitutively expressed to identify successfully transfected cells. C-to-T editing by the APOBEC-Cas9 editosome...that results in the restoration of fluorescence. Transient reporter for editing enrichment (TREE) The Wang...Wang and Brafman lab developed a transient reporter for editing enrichment (TREE) to purify single base... to cells segregated solely by a reporter of transfection, which only report the efficiency of plasmid...

...therapies to clinical trials in 2021. Figure 1: Schematic of the human MECP2 locus and MECP2 protein...References: Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY (1999) Rett syndrome is caused...collection of molecular biology tools so that both veteran and new Rett researchers can find the reagents ...maturation (Tillotson & Bird, 2019) and is a global transcriptional regulator of thousands of genes. Studies have...have suggested roles in transcriptional repression, activity dependent de-repression, chromatin remodeling...groove of AT-rich DNA sequences to regulate transcription (Baker et al., 2013). Mutations that affect ...gene editing, MECP2 reactivation, RNA editing, RNA trans-splicing, and protein replacement to reverse the...

...levels of DnaA to get the job done.   Figure 1: DNA melting by DnaA and IHF is the first step of ...the proteins cooperatively can separate the DNA strands. Created with BioRender.com. Once DnaA breaks...it recruits helicases to continue unwinding the strands. This creates two replication forks that move in...elongation phase, a DNA polymerase synthesizes a new DNA strand at each replication fork. Termination sequences...act as a feedback mechanism to reduce further transcription. Replication is also regulated by the balance... concentration is controlled at the level of translation. RepZ mRNA must fold into a “pseudoknot” held...intramolecular base pairs for the protein to be translated. An antisense RNA called Inc, also encoded by...

... over again. Here’s that process!   Figure 1: The Design-Build-Test-Learn cycle adapted for video...looking, I found an inexpensive HDMI transmitter that could transmit a signal between a wall. There was ... things first was getting that wireless HDMI transmitter/receiver, to beam the camera’s image to my remote...then set up a monitor connected to the wireless transmitter and an iPad in the office next door (connected...

...first foray into Reddit. Here are some reasons why: 1. Reddit has a large and active community of people... general advice on how to meet people, how to transition out of academia, and other science career topics.... Although I didn’t start answering until 5pm, frankly, I was typing far before five. Fortunately I have...

...gene, and a terminator, as in Figure 1). Next, up to six Level 1 modules can be assembled into a Level...directionally assembled into a Level 1 vector creating a single transcriptional unit (for example, a promoter...coli to maintain the final plasmid after transformation. Figure 1: Overview of the MoClo Toolkit components...consists of three sets of cloning vectors (Level 0, 1, and 2) which can be used in successive assembly steps... mammalian expression vectors for transient or stable transfections. PUF Assembly Kit Mammalian Expression...nucleases (TALENs) and transcription factors (TALE-TFs) for genome editing and transcription modulation applications...multigene constructs. MoClo Plant Parts III: Transformation & Genome Engineering Kit Plant Expression, ...

...cells were transfected using 1:1, 1:2, 1:3 and 1:6 µg of pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided...18.9 18.9 1:2 18.9 37.8 1:3 18.9 56.7 1:4 18.9 75.6 1:5 18.9 94.5 1:6 18.9 113.4 Gently add the diluted PEI...possible range of ratios to test: Ratio of DNA:PEI Amount of DNA (μg) Volume of 1 mg/mL PEI (μL) 1:1 18.9 .... Thawed aliquots should be discarded after 1–2 months. 1 mg/mL polyethylenimine, linear MW 25,000 Da ...stable. Dilute 1:3 (µg DNA:µg PEI) in 500 µL total of OptiPro SFM (per 10 cm plate). 56.7 µL of 1 mg/mL PEI...for viral production) Day 1: Transfect Cells Day 2 (am): 18 h post transfection - Remove media, replace ...prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1–2 days ...

...Communications , 15 (1), 3663. PMID: 38688943 Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann...pooled format into lentiviral transfer vectors (Figure 8B). Libraries come in 1-vector systems, in which Cas9..., 1163–1171. PMID: 23979020 Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D..., 17 (5), 471–479. PMID: 32203383 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I....CRISPR-Cas9. Cell , 167 (7), 1829-1838.e9. PMID: 27984730 Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott...Nature Protocols , 8 (11), 2281–2308. PMID: 24157548 Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg...Communications , 14 (1), 6175. PMID: 37794046 Small precision edits Anzalone, A. V., Randolph, P. B., Davis,...