We narrowed to 605 results for: RAN-1

...packaging transfection. In my lab we regularly use the ratios below: 1 Transfer Plasmid : 1 pHelper: 1 RepCap... plan to transfect: 1 to 10 Fill in experimental details on the spreadsheet: Name your viral prep...calculator in the comments section below. References 1. Choi, Vivian W., et al. "Production of Recombinant...prevent future headaches. To help you plan PEI transfections for AAV packaging, considering using this PEI...Calculator. AAV packaging typically requires transfecting three plasmids at specific molar ratios. To ...PEI calculator for AAV packaging Use the PEI transfection calculator for AAV packaging Select the plasmid...RepCap OR 3 Transfer Plasmid : 5 pHelper: 2 RepCap Select what type of tissue culture dish you will be ...

...type of choice in vitro is known as reprogramming [1]. The process can be divided into two stages: Dedifferentiation...//www.linkedin.com/in/kmukherjeephd/.  References 1. Hochedlinger, K. and R. Jaenisch, Nuclear reprogramming...from human peripheral blood. Cell Stem Cell, 2010. 7(1): p. 15-9. PubMed PMID: 20621044. PubMed Central PMCID...induced pluripotent stem cells. Hepatology, 2010. 51(1): p. 297-305. PubMed PMID: 19998274. PubMed Central... pluripotent stem cells. Nat Biotechnol, 2014. 32(1): p. 84-91. PubMed PMID: 24291815. PubMed Central ...keratinocyte lineage. Methods Mol Biol, 2014. 1195: p. 1-12. PubMed PMID: 24510784. PubMed Central PMCID: PMC4096605...induced pluripotent stem cells. PLoS One, 2010. 5(1): p. e8763. PubMed PMID: 20098701. 31. Tang, Z.H., et ...

...details on how they work!) Figure 1: Base transitions are edits between the purine bases (adenine... inducing targeted DNA transversions in human cells. Nature Biotechnology, 39(1), 41–46. https://doi.org...R-loop structure. In order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and...given the pithier name CGBE1, for “C → G Base Editor 1,” and had a C → G editing efficiency of up to 68%... the CGBE: DNA polymerase D2 (POLD2), exonuclease 1 (EXO1), and RNA binding motif protein X-linked (RBMX...excision repair proteins. Nature Communications, 12(1), 1384. https://doi.org/10.1038/s41467-021-21559-9...machine learning methods. Nature Communications, 12(1), 4902. https://doi.org/10.1038/s41467-021-25217-y...

...activity in single cells using translocation reporters. Nature protocols, 13 1, 155-169. PMID: 29266096. Find...Kinases: they regulate many proteins, with ~1/3 of human proteins predicted to be phosphorylated on at...which have previously been characterized. See Box 1 in Kudo et al. for more details on the design of the...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...activity is enhanced, leading to cytoplasmic translocation of the reporter. As a result, the KTR is shuttled...

...Table 1 has a detailed list of the bacterial species these plasmids are compatible with.  Table 1: Non-...Figure 1: Idealized examples of a narrow host range plasmid with only one ori and a broad host range plasmid...Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria. Gene, 70(1), 191–197. https... N.J.), 765, 327–343. https://doi.org/10.1007/978-1-61779-197-0_19  Meyer, R. (2009). Replication and ...?  Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Tranfection...define plasmid host range, identify a few of the features that confer broad host range in plasmids in nature...features to create broad host range recombinant vectors.  A plasmid’s host range is determined by the different...

...138920210790886871 Pray, L. (2008) Transposons: The jumping genes. Nature Education 1(1):204. https://www.nature.com...reverse transcriptase activities.    Figure 1: Overview of retrotransposon transposition. Retrotransposons...are also known as retrotransposons. They transpose by a ‘copy-paste’ mechanism (Fig. 1). They first copy...their host’s genome.   There are two types of Class 1 TEs: those with long terminal repeats (LTRs) and those...CRISPR-Cas system. This system has four major components: 1) a CRISPR RNA (crRNA), 2) four proteins (TniQ, Cas8...express a transposase or reverse transcriptase so they can transpose. Both class I and II transposons can be...eukaryotic genomes.  Components of a transposon system Transposon: The transposon is the DNA sequence that moves...

... compounds with various antimicrobial activities (1). She has developed a fast endophyte screening system...us coordinate and promote the awards. References 1. Martinez-Klimova, Elena et al. “Endophytes as sources...antibiotic” Biochemical Pharmacology 2017 Jun 15;134:1-17. PubMed PMID: 27984002. 2. Bilan, Dmitry S. et ...Addgene-Seeding Labs Plasmid Grant Check Out Previous Addgene-Seeding Labs Plasmid Grant Awardees Listen to Our...Labs and Addgene. The Addgene-Seeding Labs plasmid grant is a collaborative endeavor providing plasmids to...who were awarded the Addgene-Seeding Labs Plasmid grant this year. These scientists work on projects spanning...and to increase the potential for this drought tolerant plant, Dr. Robertson aims to make use of the CRISPR...

...each reporter or check out table 1 for a summary. HRP HRP tagged RANbodies don’t require a secondary antibody... Table 1: Summary of the Key Features of HRP, Spaghetti Monster, and Chicken IgY Domain RANbody Reporters...NIH’s Developmental Studies Hybridoma Bank (DSHB). 1 ml of antibody-containing hybridoma supernatant is... generate and costly to share. What is a RANbody? A RANbody is a fusion between a reporter and a nanobody...nanobody sequence could be used to make a RANbody. RANbodies are expressed in 293T cells and are secreted...smaller size of RANbodies compared to antibodies. Staining with multiple HRP-RANbodies is indistinguishable...HRP-conjugated RANbody can be used at a time. Samples can be sequentially stained with two HRP RANbodies if the...

...course much more difficult than in cell culture, but Ran et al. have shown that CRISPR and AAV can be used...Thakore PI, Moreb EA, Rivera RM, Madhavan S, Pan X, Ran FA, Yan WX, Asokan A, Zhang F, Duan D, Gersbach CA...WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, ...: 25692716. PubMed Central PMCID: PMC4335351. 9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...X-linked recessive disorder affecting approximately 1 in 5000 males. DMD is caused primarily by frameshift... applicable to 80% of DMD patients. Figure 1: Exon skipping strategies for Duchenne muscular dystrophy...needed. First, CRISPR delivery must be optimized to: 1. Reach a high percentage of muscle cells throughout...

...obstacles when working with virus: 1. Viral vectors undergo DNA rearrangements. Depending on the composition...approximately 20–50% of the packaging cells after 1–3 days of selection. A lower percentage of surviving...up-to-date tips for your virus experiments. References 1. Anthony, Simon J., et al. "A strategy to estimate...may undergo genomic rearrangements during replication. To avoid genomic rearrangements, try amplifying the...immediately prior to their transduction has been shown to increase AAV transduction efficiency by approximately...immediately following transfection. In other words, when exchanging the medium after transfecting cells, replace...Fibronectin is another membrane-interacting protein that can be used to enhance transduction efficiency in cells...

...if you obtained a transformant with a single insertion because it will segregate 3:1 in the T2 generation...lineage and has made countless transgenic plants in the process.     References 1. Emami, S., Yee, M. and Dinneny...Methods. BioMed Central, 2(1), p. 19. doi: 10.1186/1746-4811-2-19. PubMed PMID: 17087829. PubMed Central... which is ease of transformation. There are many motivations to generate transgenic Arabidopsis, from ...from studying transcriptional and translational dynamics of genes and proteins in living plants, to complementing... dip transformation method. The general steps for this method include: Cloning and transforming a plasmid...integrates transfer DNA (tDNA) into the genomes of the plants it attacks Growing the transformed agrobacterium...

...focus on the AAV transfer plasmid and take a look at each of its parts. Figure 1: Parts of an AAV...human synapsin 1 promoter is neuronal specific (Haery et al., 2019). Most AAV transfer plasmids have a...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...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...

...Check out Addgene's New Viral Service! References 1. Hay, R.J., Macy, M.L. & Chen, T.R. “Mycoplasma infection...contaminations.” Methods in Molecular Biology 946 (2013). 1-13. Pubmed PMID: 23179822. 5. Segura, M.M., Kamen,...Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors."Nature protocols 4.4 (2009...may not translate well to other cell types. To overcome this issue, try transducing with a range of dilutions...DEAE-dextran can improve transduction efficiency. In addition, this study found that transduction efficiency...passage number of a culture the lower the transfection and transduction efficiencies. Cultures should only be...others opt for a single harvest after transfecting with the transfer and packaging plasmids. A benefit of...

...regulated in the medicinal plant, the Catharanthus roseus (Figure 1). The ultimate goal of our research ...reports 9.1 (1990): 1-5. PubMed PMID: 24226366. Lee, Min Woo, and Yinong Yang. "Transient expression assay... plant scientists do transient leaf transformations The development of transgenic plants is a common problem...that the transformation and gene expression can be tracked. During a transient leaf transformation, the engineered...method. Transfer your plant into the dark 16 hours before the transformation. We routinely transfer our plants...developing transgenic hairy root lines in tissue cultures takes half a year, and generating a transgenic plant...plant can take even longer, a transient plant leaf transformation process could save the plant biologist...

...overcomes many of those limitations (Figure 1). Figure 1: Opto-katanin induces fast, localized microtubule...Signaling Toolbox. ACS Synthetic Biology, 11(1), 515–521. https://doi.org/10.1021/acssynbio.1c00604.  Mahlandt...specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized iLID along with...cell membrane isn’t powerful enough, what about altering the chemical composition of the membrane itself...SuperPLD hydrolyzes membrane PC to PA. A) OptoPLD is recruited to plasma membrane or lysosomes and its...optogenetic tools to control the cytoskeleton and cell membrane dynamics. First, a quick refresher.  The tools...undergoes a conformational change and binds the plasma membrane under blue light CRY2-CIB1 heterodimerizes or ...

...glossary at the end of this post! Figure 1: Base transitions are edits between the purine bases (adenine...uracil DNA glycosylase. Nature Communications, 15(1), 4897. https://doi.org/10.1038/s41467-024-49343-5...uracil-DNA glycosylase. Nature Communications, 15(1), 6397. https://doi.org/10.1038/s41467-024-50012-w... the non-edited strand to encourage the cell to repair the DNA using the edited strand as a template —...editing. Like other transversion editors, this editor had relatively high indel rates ranging from 4.7% to 30%...our last post, we talked about the first base transversion editors: CGBEs, or C → G Base Editors. CGBEs...editor could excise other bases? Could other transversion editors be close at hand? Once again, this post...

...whether they could 1) be decorated with proteins and remain functional, 2) break membranes outside of their...Follow her on Twitter @jessicapolka.     References 1. Kanabrocki, J. A., Quackenbush, R. L. & Pond, F. ...learn a lot from Nature’s simpler solutions to transmitting forces over long distances. For instance, how..., R bodies do not exert force by dynamically rearranging into different polymerization states. Instead...demonstrates that R bodies can function to break membranes of different organisms. And finally, we demonstrated...could use them to lyse cells (and surrounding membranes) in keeping with their natural function (see below...

...Addgene, we use seven different types of “plates” (Fig. 1). When you have to tell an engineer that you want ...”? We took steps towards solving this problem by 1) making sure we qualified these words when we discussed...resources on the Addgene blog Read about other transferable skills Find all of our science career related...

...offers insight into a career as a Professor. Step 1: Your PI forwards you a Request For Applications (...proposal typically lists the deliverables, explains 1-2 potential pitfalls of the intended protocol, and...improve your grant writing skills. Good luck! Further advice on grant writing Since successful grant writing... during the grant application process. When my PhD advisor first asked me to write a grant application... advice on writing a grant proposal. "Murder Most Foul: How Not to Kill a Grant Application" by Vid Mohan-Ram...their advisor alone applies for these competitive grant applications; however, in some cases, your advisor...advisor may choose to allot some if not all grant duties to trusted students. For example, if the proposal...

...plasma HIV-1, improving life expectancy and quality for HIV-1 patients. However, ART is not an HIV-1 cure. ...activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that can produce HIV-1 toxic...with HIV-1. For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy number...HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 to remove the HIV-1 genome...currently infected with the lentivirus HIV-1. Today, HIV-1 can be controlled with antiviral therapies ...take on HIV-1 in vitro, and what obstacles must be overcome for clinical success. ART and HIV-1 reservoirs...Cutting HIV-1 out of the genome with CRISPR/Cas9 Before the introduction of CRISPR, HIV-1 genome editing...

...improve your grant writing and hopefully increase your success rate. 1. Submit your grant early! First...involved so it’s important not to throw in the towel if 1 or 2 funding bodies say no. If possible, get feedback...in writing small travel grants to attend conferences and a seed funding grant for a proof-of-principal...www.assaygenie.com. No matter what facet of academia you are in, grant writing can be the lynch pin that results in your...to be much use. 2. Originality is essential for grant success The two most important things reviewers ...sure that comes across. Check out some in-depth grant writing tips at the Human Frontier Science Program's...Skills 5. Practice makes perfect, apply for small grants too There are actually quite a lot of different...

...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems...archaea, though Class 1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible...optimal plasmids to harness the power of Class 1 systems. Class 1 CRISPR systems can be further broken down...6105. https://doi.org/10.1093/nar/gku241 Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N....types, 33 subtypes, and 17 variants. Figure 1: Overview of the CRISPR classification hierarchy. ...effector complex after binding to a target. Class 1 is the most abundant of the two, comprising about ...incredible abundance, Class 1 CRISPR systems are less popular in the lab. This can be attributed to the multi-Cas...

...crevices proteins on the cell surface. Figure 1: Comparison of common antibody, HCab, and nanobody...Humana Press, pp 133–148. https://doi.org/10.1007/978-1-62703-293-3_10 Walter J et al. (2020) Synthetic nanobodies...sybodies against membrane proteins Developing antibodies against proteins embedded in membranes is tricky because...develop antibodies against unstable targets like membrane proteins that may unfold due to camelid high body...synthetic nanobodies, called sybodies, against membrane protein targets in as little as 3 weeks (Zimmermann...lab can quickly select for sybodies targeting a membrane protein of interest using the Sybody Generation...have a concave, loop, or convex scaffold and are randomized with a defined set of different trinucleotides...

...phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably T4 DNA ligase) are ... enzymology of DNA repair enzymes.     References 1. Shuman, Stewart, and Christopher D. Lima. "The polynucleotide...thermostable DNA ligase-encoding gene." Gene 109.1 (1991): 1-11. PubMed PMID: 1756968. 7. Lauer, G., et al. "Cloning...19329793. PubMed Central PMCID: PMC2719376. 5. Barany, Francis. "Genetic disease detection and DNA amplification...1986365. PubMed Central PMCID: PMC50775. 6. Barany, Francis, and David H. Gelfand. "Cloning, overexpression...Central PMCID: PMC208194. 8. Luo, Jianying, and Francis Barany. "Identification of essential residues in Thermus...(1989): 245-254. PubMed PMID: 2753355. 12. Barany, Francis. "The ligase chain reaction in a PCR world....

...expressing additional EBNA-1 transiently. The transiently expressed EBNA-1 increases protein expression...are based on the Epstein-Barr Nuclear Antigen-1 (oriP-EBNA-1) system where each vector contains a viral ...replication, OriP and the EBNA-1 sequence that encodes for the DNA binding protein EBNA-1 [7]. These two sequences...extra plasmid, pCXWB-EBNA-1 is used in the reprogramming cocktail. pCXWB-EBNA-1 lacks oriP and is unable...pCXLE vectors listed below in Table 1 are available at Addgene. Table 1: List of pCXLE episomal plasmids,... and shRNA against p53 pCXWB-EBNA1 37624 transient EBNA-1   Many thanks to our guest blogger, Kusumika...obtained and the subsequent reprogramming success [1]. Fibroblasts need to be cultured and expanded in ...

...between strand 10 and 11 to yield large (1–10) and small (11) fragments. In several cases an FP(1–10) fragment...General Design Figure 1: Two proteins are fused to the FP(11) and FP(1–10) fragments. When Protein... to Plasmids) PI Blue EBFP2 EBFP2(1–10) and Capri(1–10) for use with GFP(11) Multiplexed labeling...Commun. 2017 Bo Huang mNeonGreen3 mNG3K(1–10) and mNG3A(1–10) for use with mNG2(11) Improved yellow-...sfCherry2 sfCherry2(1–10) and sfCherry2(11), also photo-activatable variant PAsfCherry2(1–10) Improved split...brings the pieces of the FP back together (Figure 1). This method is generally known as Bimolecular Fluorescence...even from a different base FP (for example, EBFP2(1–10) can be used with GFP(11)). Some of the articles...

...probably have better specificity. A good reference is Ran et al. (2013). As for target selection, we usually...fine based on new data). For general transfection, you could use 1 µl of 10 µM oligo stock for each well...well in 24-well plate. A good example/reference is Ran et al. (2013). In the case where you have intact...https://doi.org/10.1534/g3.112.005439  Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N.... from Double-Strand Break Repair in Mammalian Cells. Molecular and Cellular Biology, 18(1), 93–101. https...https://doi.org/10.1128/mcb.18.1.93  Ran, F. A., Hsu, P. D., Lin, C., Gootenberg, J. S., Konermann, ...1389. https://doi.org/10.1016/j.cell.2013.08.021  Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott...

...making it easier for us to do our work.” References 1. Athanasios Lampropoulos et al. "GreenGate - A Novel...terminators and resistance cassettes for selection of transgenic plants. The complete GreenGate toolkit, now available...the time required to generate multi-construct transgenic plants. In the most practical terms, what GreenGate...Versatile and Efficient Cloning System for Plant Transgenesis." PLoS ONE. 8(12): e83043 (20 December 2013)...

...just make their own buffers when the kits buffers ran out. Homemade buffers performed the same as those...series to increase binding capacity. Figure 1: Purify DNA using glass filters. Image: Kim and Morrison...a gravity-based column plasmid maxiprep kit takes 1-1.5 hours to complete, while this syringe-based method... solution. NaI has two purposes in this protocol: 1) it solubilizes both DNA and agarose, and 2) it helps...property and developed a protocol that uses Whatman No. 1 paper or even paper towels to purify DNA and RNA from...rather a glass powder slurry that has a milk-like appearance. To make DNA stick to the glass particles in ...the Patterson Protocol was shown to purify DNA ranging in size from 100 bp to 48 kb with high yields and...

...    Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32. Each individual...series that ranges from 1:2–1:32. The specific dilution series used will depend on the expected concentration...the data points fit the trendline. R2 ranges from 0 to 1 with 1 being a perfect fit and 0 indicating that...example, Figure 1 depicts a 2-fold dilution series. Each step of the series dilutes by 1:2 for a series...individual step of the series is consistent at 1:2. When preparing a dilution series, use a new pipette tip for...concentration lies within the dynamic range of the curve. The dynamic range is the linear span between the lowest...the dynamic range of your standard curve, so if your sample does not fall within this range, it may be ...

...cells transduced with lentiCas9-blast. A549 cells were transduced (MOI = 37) and selected with 1 µg/mL...expressing Cas9 via transduction with Cas9 lentiviral vector, lentiCas9-Blast (52962-LV, Figure 1). Some cells...were frozen down. Figure 1: Cas9 expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9...of the gel in the BRAF gRNA-transduced samples (data not shown). Pro-Tips: 1.Designing primers against ...The entire process from start to finish took about 1 month. Cas9 delivery Overview: Before any editing...Western blotting is a loading control. Pro-Tips: 1. It’s all about the MOI. In retrospect I used a way... pool had wildly variable Cas9 expression (Figure 1). This is a problem because Cas9 expression affects...

...). Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and...Overview. Cells, 12(5), 1–41. https://doi.org/10.3390/cells12050785 Murlidharan, G., Samulski, R. J., &... ONE, 9(8), 1–10. https://doi.org/10.1371/journal.pone.0106472 de Backer, M. W. A., Brans, M. A. D., Luijendijk...vector-mediated transduction of the corticospinal tract: Comparison of four promoters. Gene Therapy, 28(1–2), 56...the selection process. Factors to think about are (1) your experimental conditions and measurements; (2...serotype, promoter, and titer.     Figure 1: Flow chart depicting a) steps for selecting serotypes...injection site than AAV2 (Watakabe et al., 2015) (Figure 1). You’ll need to consider if such properties could...

... Table 1: Transduction efficiency of AAV-PHP.B versus AAV-PHP.eB in the CNS. A GFP transgene was packaged...marker). Check out tables 1 and 2 below for a summary of the quantified transduction efficiency of AAV-PHP.eB... AAV.PHP.S’s great neuronal transduction efficiency, as shown in tables 1 and 2, with a cell-type specific...nervous system (CNS). AAVs are popular tools because: 1) their genomes are easy to manipulate, 2) they have... AAV vectors. What can these AAV’s be used for?  1. Multi-color labeling of neurons Multi-color labeling... dendrites extend to. The keys to this method are 1) high levels of color diversity so that cells are ...expression into a two part system (figure 4d):  Part 1: A cocktail of three AAV vectors expressing RGB proteins...

...outside of the antibody’s dynamic range. Specifically, the 1:2, 1:1, and No dilution samples all have ...dilution range from 1:100–1:1,000. If your antibodies come from ascites fluid, try a dilution range from ...with a recommended dilution and/or range (typically between 1:500 and 1:10,000 for primary antibodies). ...from 1:1,000–1:100,000. Secondary antibodies For secondary antibodies, the recommended dilution range is ...is usually between 1:5,000 and 1:200,000. Check the manufacturer’s recommendations, and if needed, optimize...marked on the membrane (1) using the gel as a guide. The gel is then removed from the membrane (2) and the...Mark your membrane! Membranes look the same from both sides, so it’s quite easy to think lane 1 is‌ lane...

... assembly reaction** 1 5 min., 37 ºC (2.5 μl) 687 100% 274,200 2,742,000 1 60 min., 37 ºC (2.5 μl)... volume of the 1 ml outgrowth spread on each transformation plate can be manipulated to result in appropriate...Figure 3, Table 1). These improvements (5- to 12-fold increases in the number of transformants while maintaining...expected complete sequence for the lacI/lacZ genes (1), while sequencing of white colonies showed a mixture... obtained from 1-, 12- and 24-fragment assemblies of the lacI/lacZ cassette, and illustrates how the volume...fidelity studies conducted by Potapov et al. at NEB (1, 2). While T4 DNA Ligase, the mainstay of most biotechnological... 16 ºC) x 30 (100 μl) 78 90.7% 783 9,792 Table 1. Yields and fidelities for Golden Gate Assemblies ...

...Table 1 below outlines a few of the more common genetic changes found in E. coli strains. Table 1: Common...amber (UAG) stop codon by tyrosine insertion λ-thi-1 or thi1 Mutation in thiamine metabolism Requires exogenous... all based on E. coli K-12 and are considered BSL-1. Table 2: Lab strains of E. coli Strain Natural...plasmids, blue/white screening. F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF...) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ- JM109   General cloning and...strain for cloning repetitive DNA. endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ Δ(lac-proAB) e14- [F' traD36...of an E. coli K-12 strain. F- λ- ilvG- rfb-50 rph-1 NEB Stable   For cloning into and storage of lentiviral...

...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the ...sequence of the translated protein.  These changes can be 5’ or 3’ of the desired change.   Table 1: Overview...recombineering with an ssDNA oligo substrate.   Figure 1: Components of the Lambda Red Recombineering system...and tags.  The typical frequency of recombinants is 1 positive clone out of 104 to 105 colonies. Pro Tip... with a recombination frequency between 0.1% to 1%, and can be increased to as high as 25-50% by designing...replication. There are two ways to avoid activating MMR: 1) use a strain of bacteria that has key MMR proteins...

...They help us to transform raw genomic coordinates into meaningful experiments. Table 1: An emerging family... chromosome, start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning...genetic elements like protein, RNA, and DNA (Table 1). As with GPS in the real world, coordinates alone...enhancers with the help of genome math. Figure 1: Genome math illustrated. Intersect (left) and set...histones with “GPS for proteins,” ChIP-seq from Table 1. Because of DNA’s 3D geometry and the chemical properties...experiment to identify the critical NANOG binding sites: 1. Find Enhancers that have NANOG binding sites 2. Design... In order to denote the NANOG enhancers from step 1 with genome math, we’ll need a bit of shorthand from...

...like uneven pipetting or irregular membrane transfer. Figure 1: A loading control is a type of positive...because the loading control band is weaker in condition 1 than condition 2, the normalized values for the two...to using loading controls accurately. Assumption #1: The loading control is not perturbed by the experiment...without your knowledge? If you can’t trust Assumption #1, any change in the normalized intensity of your POI...interest band is significantly lighter in “condition 1” than “condition 2.” By eye, the loading control appears... the protein abundance axis, both blue “condition 1” points (POI and loading control) are smaller than...assumptions discussed above into question. Assumption #1: The loading control is not perturbed by the experiment...

...Inactivation of AAV Serotype 1 Vectors. Human Gene Therapy Methods, 28(1), Article 1. https://doi.org/10.1089...commonly called “triple plasmid transfection” (Figure 1).    Figure 1: Overview of key steps of AAV production...repeat sequences. Human Gene Therapy Methods, 23(1), Article 1. https://doi.org/10.1089/hgtb.2011.034 Benyamini...in the CNS. Molecular Neurodegeneration, 15(1), Article 1. https://doi.org/10.1186/s13024-020-00361-z ..., 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...cells happy. Harvesting virus Once you have transfected your cells, wait 2–5 days and then collect the supernatant...

...doi.org/10.1186/s13059-018-1591-1 Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang... 556:57–63 . https://doi.org/10.1038/nature26155 Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz... modify any desired genomic target provided that (1) the sequence is unique compared to the rest of the...will cover three ways to circumvent this limitation: 1) the use of novel S. pyogenes Cas9 variants with varying...Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged...SpCas9 variants effectively double the targeting range of CRISPR/Cas9 within the human genome. Another ...known to produce off-target editing with SpCas9 (Amrani et al., 2018). When choosing a Cas9, remember to...

...loops 1 and 6 resulted in light controlled binding, but with opposite effects: insertion into loop 1 caused...experiments.  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced...favorite protein (YFP). You have two options:  Option #1: Affinity tag purification You tag YFP and use an ...and one against GFP. Insertion of AsLOV2 into loop 1 always generated OptoNBs with dark-induced binding... induced dark-induced blue-light induced Table 1: Summary of the effect of AsLOV2 insertion site on...let the team detect its co-localization with a membrane-localized mCherry or GFP protein. With this system...bind, it co-localizes with its target at the cell membrane and when the OptoNB can’t bind, it localizes to...

...in the immune system. Nature Immunology, 25(1), Article 1. https://doi.org/10.1038/s41590-023-01689-6 ...expression vectors (Table 1). The four systems Name Function Plasmid Transduction Marker Mouse Strain... GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME systems Proof of Concept These systems were...was lethal (LaFleur et al., 2024).   Figure 1: Schematic showing the X-CHIME approach. Figure from...genes in the immune system. Nature Communications, 10(1), 1668. https://doi.org/10.1038/s41467-019-09656-2... LaFleur et al., 2019b). CHIME works through transduction of Cas9-expressing (Rosa26-Cas9) hematopoietic... a lentiviral vector containing a gRNA. These transduced stem cells can be implanted in irradiated recipients...

...2017. 1. Among scientists, historians have the most Twitter users. Biologists rank #8. Figure 1: Highly...that these results could be biased for two reasons: 1) they are based on the US Occupational Employment ...female to male = 0.629 (the closer this ratio is to 1, the closer males and females are to parity - the ... authorship in the United States (0.428). 4. The #1 (re)tweeted domain by scientists was Instagram Social...links to research that isn’t behind paywalls. 5. The #1 (re)tweeted science domain by scientists was Nature...Discipline. Biologists, neuroscientists, and ecologists (orange box) post tweets containing scientific domains ...network of life (green), physical (blue) and social (orange)  scientists largely cluster by discipline, suggesting...

...was around 33% of that observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation...paper. Green and red lines depict the sum of mean +1 and -1 std for each paper, respectively. The fitted...complication in the China data – there is a period of 1 month, from 1/22/20–2/23/20, where there were zero plasmid... from 5/22/14 to 5/19/20). Each paper has between 1–32 plasmids and a total of 2–2,590 requests. We received...correspond to publications of popular papers. Figure 1: Plasmid requests by date. Parentheses depict the ...China. However, it is evident that if we believe that 1/22/20–2/23/20 are the more accurate dates of lockdown...cases being reported (right, red), likely due to quarantining {% video_player "embed_player" overrideable...

...assembly. Journal of biological engineering. 2010;4(1):1. Pubmed PMID: 20205762. PubMed Central PMCID: PMC2822740...with minimal spectral overlap between them (Table 1). In each construct, 3 copies of the fluorescent protein... enabled labeling of particular structures (Table 1). Each construct was flanked with a CMV promoter (...construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling, with...fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave researchers the ability ... all four constructs into one 15 kb insert. Table 1: MXS Constructs for Labeling Cellular Structures ...maxima Tethering partner Subcellular localization 1 TagBFP 399nm/ 456nm histone 2B (H2B) Chromatin 2...

...endogenous gene expression (2). Our paper (1) explains how to arrange attB-attP pairs on a register to record...recombinase-based state machines in living cells” (1), into a more straight-forward, concise explanation...an RSM is to sequence the register. In our paper (1), we allowed individual bacterial cells to form colonies...in the Materials and Methods section of our paper (1). Lastly, it is possible to build gene regulatory ... not. How might one implement RSMs? In our study (1), we implement RSMs in E. coli with two plasmids (...performance data from the indicated figures in our paper (1)) Input Plasmid Output Plasmid Fig. 3A pNR64 pNR160...in the Material and methods section of our paper (1). How might one troubleshoot GSRMs that don't work...

...used in step 1. Then this strand is degraded with ExoIII and ExoI. Synthesize 2nd mutant strand: The second...are the same sense as the strand of DNA degraded in the first digest (step 1 in above diagram). Remember...making some or all of the following modifications: 1) increased concentration of Taq polymerase, 2) increased...introduced, and scalability (see Supplementary Table 1 of Wrenbeck et al). It uses a uracil labeled wild-...primers to introduce all desired codon changes (step 1 in the image above). A second universal primer is ...number of positive clones. See Supplementary Note 1 in Wrenbeck et al for details on how nicking was optimized...time no, but there are 2 instances where it does. 1) When the backbone vector has multiple BbvCI sites...

...references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid is identified, re-transform into a bacterial...rAdV-S). HEK293 cells are transfected with the AdV plasmid construct from step #1 and allowed to stay in ... (ITR) present at both ends.   Figure 1: A) The structure of an adenovirus: the virion is ~ 100nm... 5 key steps: Construction of the rAdV plasmid (~1 week): The AdEasyTM system is the most popular method... it will result in very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more HEK293...-4 rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification should result...CsCl are commercially available. Titer calculation (1 or 10 days) Physical titer (particle count, rAdV ...

... Oligos Oligo 1: Add 'tcaa' to the 5′ end of your forward guide sequence (see Figure 1). Oligo 2: Add... Figure 1). Perform standard phosphorylation and annealing of the two oligos. Figure 1: Components... promoter for ωRNA (reRNA) expression (see Figure 1). Both plasmids contain a hygromycin resistance gene...provide a step-by-step guide for using a Dra2TnpB. Step 1: Design the 20-nt Guide Sequence Identify a TAM site...protocols (pp. 245–260). https://doi.org/10.1007/978-1-0716-4358-7_20  Karmakar, S., Panda, D., Panda, S... induction of meristems. Nature Biotechnology, 38(1), 84–89. https://doi.org/10.1038/s41587-019-0337-2...1166–1187. https://doi.org/10.1038/s41477-021-00991-1  Qiao, J., Zang, Y., Gao, Q., Liu, S., Zhang, X.,...