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...118). Humana Press. https://doi.org/10.1385/1-59259-301-1:99 Resources on Addgene.org Addgene antibodies...antibody detection is desired… and yes, we have an antibody for that!   Fig. 1 - Diagram of His-tagged...You’ve designed the perfect experiment – controls, conditions, and everything in between – now all you...This is where affinity tags come in – they are peptides which do not naturally exist in most expression...protein for downstream applications Below, we describe a few of the most common tags available. Maltose...antibody is an alternative to amylose resin and also provides a visual detection mechanism via applications ...

...collected from heterozygous individuals. Only 1/4 of the descendants of the heterozygous plants, therefore, ...in all other plants, anthocyanins. Figure 1: An adult plant expressing RUBY. Photos by Andrew ... (2020). https://doi.org/10.1038/s41438-020-00390-1 Additional resources on the Addgene blog  CRISPR ... Antibiotic selection would only then kill the desired plants! PCR or fluorescent markers are currently... have similar drawbacks, often requiring the destruction of plants or tissue for GUS staining or are not...from these plants will be green, containing the desired mutation but lacking both RUBY and the construct...compared to other markers lends itself well to nondestructive selection. If RUBY was integrated into a CRISPR...

...occur. While the Brainbow-1 system employs Cre-mediated deletion, the design of Brainbow-2.1 cleverly ...homologous, preventing design of antibodies specific to each fluorophore. Third, Brainbow-1 and Brainbow-2 each...the Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow...dendritic processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...Rheenen J, Simons BD, Clevers H. Cell. 2010 Oct 1;143(1):134-44. PubMed. Drosophila Brainbow: a recombinase-based...R26R-Confetti described in Hugo J. Snippert, et al. (2010) and the MAGIC Marker strategy described in Karine...immediately following the promoter. The STOP cassette includes a mutant YFP that does not fluoresce, but can ...

...detector for that fluorophore. For example, in figure 1, the FITC signal is spilling over into the primary...spectra to use for compensation.     Figure 1: The emission spectra and filters for FITC and PE ...in the panel. This complexity index ranges from 0-1, with a value of “0” indicating those markers are ...completely distinct from one another, and a value of “1” indicating those markers are identical and completely...positive and negative populations, Careful panel design In both conventional and spectral flow cytometry...indistinguishable. This tool is of particular value when designing a panel.     Figure 3: Similarity index...if markers are co-expressed in your cells when designing your panel. If you have two highly overlapping...

...amounts of high quality DNA. Figure 1 illustrates the three grades of DNA you may need based on their ... kits are designed to accommodate specific volume ranges of culture as their input (Table 1). When you...miniprep kit for example. These kits are designed to handle ~1–5 mL of liquid culture. Using a higher volume...their relative quality and quantity.   Figure 1: Plasmid preps fall into different categories based...beads and a suitable magnet as you scale up. Table 1: Standard Prep Kit Capacities* Prep size Amount...Amount of culture Approximate DNA yield** Miniprep 1–5 mL 5–50 µg Midiprep 25–100 mL 20–200 µg Maxiprep...components (i.e. buffers, columns, or beads) are designed to work together — no mixing and matching!   ...

.... Table 1 provides an overview of pooled library protocols you can use as a reference. Table 1: Examples... be more difficult to transform.    Figure 1: Factors to consider when creating a pooled library...etc. The DNA amount can be anywhere from 10 ng to 1 μg of DNA. It often correlates with the size of the...(Are the inserts of different sizes?), and the desired coverage for the library (How important is it that...  The most important factor to consider is your desired library coverage, since this will help decide what...should always experimentally test the protocol you design to ensure that it works for your library before...Addgene. If you have any specific questions about designing your library amplification protocol, feel free...

...attract fish for them to prey upon (Figure 1). Figure 1: The sea anemone's red color can be used ...synthetic biology. Journal of Biological Engineering, 12(1). https://doi.org/10.1186/s13036-018-0100-0 Murakoshi...expression in living cells. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-48604-4 Salih...fluorescent proteins, it’s important to note that despite their distinction by name, chromoproteins are actually...chromoproteins have a shorter history. In 1987, scientists described an unusual blue-colored protein from the jellyfish... chromoprotein plasmid that combines the most desirable features: intense color, fast maturation, and ...recombination events: The plasmid carrying the desired genetic modification integrates into the genome...

...on-target and off-target editing efficiency Figure 1: Effects of (A) double and (B) single base pair gRNA-target...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...fits into the genome editing landscape. References 1. Kim D, Kim J, Hur JK, Been KW, Yoon SH, Kim JS. Genome-wide...for Cas9 (42 nt vs 100 nt.) However, the newly described non-CRISPR protein NgAgo is smaller than any of... of these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of the CRISPR nucleases...to examine Cpf1 off-target editing. First, they designed gRNAs with single and double mismatches throughout...

...against the limitations of the assay.      Fig. 1: A schematic representation of Sybr Green chemistry...Methods Mol Biol. 2018;1768:143-160. doi: 10.1007/978-1-4939-7778-9_9. PMID: 29717442. Arya M, Shergill IS...amplification of Plasmodium falciparum multidrug resistance-1 gene (pfmdr1). J Parasitol. 2011 Oct;97(5):939-42....gamma receptor genes FCGR3A and FCGR3B. PloS one, 10(1), e0116791. https://doi.org/10.1371/journal.pone.0116791...results can be affected by DNA quality and primer design, particularly for amplicons under 100 bp (Ma, 2015... concern, look for the lowest-cost option that provides you confidence in the results you need.  Whichever...by running the product on a gel. And only ddPCR provides absolute quantitative data, instead of relative...

... vectors are: 1) the potential generation of replication competent virus [usually HIV-1]; and 2) the potential...systems have been developed over the years based on HIV-1; however, the 1st-generation vectors are never used...which has been shown to repress expression of HIV-1 LTRs (more information is available here). 2nd vs...those seen when using a plasmid backbone that was designed specifically for transient expression (find some...expression of viral constructs is a useful tool as it provides a means to quickly check that a construct is functional...3rd-generation transfer plasmids have a chimeric 5'LTR that includes a CMV or RSV promoter as well as a portion of ...those three genes plus tat. The envelope plasmid provides a heterologous envelope for pseudotyping and is...

...reporter gene, which replaces the targeted gene and provides a way to track successfully engineered cells. ...genotype each animal to identify the ones with the desired genotype. Inducible and conditional systems Conditional...

...setting out of the lab (coffee, lunch or beer) and in 1:1 conversations. This creates a level of familiarity... other posts in our Science Mentors series: Part 1: "What Makes a Good Mentor?" and 6 More FAQs About...PDF at the end of this blog post. This document includes a list of questions to ask your potential advisors...some good pre-work you can find a lab that will deserve your hard work and an advisor that will be a lifelong...

...2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins. The goal of both groups was to create... the Sata lab is composed of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein...combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal fragment.... crassa) (Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and...fold-induction of Cas9 activity (16.4-fold). This paCas9-1 light-inducible system was able to recognize the same...flCas9). When triggered by blue light (470nm), paCas9-1 induced indel mutations via NHEJ (frequency of 20.5%...background activity of the system by modifying paCas9-1 using nMagC714 instead of nMagHigh1-C714, generating...

...leading professors for their top 3 tips. Tom Ellis 1. Keep up with the literature. He recommends setting...research and with getting published.” Connie Cepko 1. Work hard. There will be periods when opportunities...time comes to work with full force. George Church 1. “Initial and sustained motivation is a huge component...entertainment, politics, K-12, etc.” Addgene's team includes many PhD scientists who have stepped away from...

...by mating with Cre-expressing mice. Figure 1: (A) Schematic of the Cre-dependent SPH construct ... alpha (aCaMKII; excitatory neurons) and Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can...ubiquitous expression of sgRNA(s) and mCherry (vector 1) with neuron-specific expression of dCas9-VPR (Figure...Neuron-specific expression is mediated by the synapsin 1 (SYN) promoter, which was superior in its ability ...Patrick Miller-Rhodes from the University of Rochester Medical Center. Patrick Miller-Rhodes is currently...This post was contributed by Patrick Miller-Rhodes, a Ruth L. Kirschstein NRSA Predoctoral Fellow at ...this technological gap, a trio of recent papers describe the development of CRISPR activation (CRISPRa)...

...’s fused to the C-terminus of DsRed.T3. See Table 1 for a summary of Rosella’s excitation and emission...fluorescence Rosella emits based on its localization. Table 1: Summary of Rosella’s excitation and emission spectra... variants are available from Addgene. References 1. Rosado, C., Mijaljica, D., Hatzinisiriou, I., Prescott...single fluorescent biosensor. Scientific Reports,5(1). doi:10.1038/srep12397. PubMed PMID: 26215030. PubMed... phosphatase activity: In yeast, the PHO8 gene encodes a vacuolar alkaline phosphatase. Normally PHO8 ...

...total dose between 1 x 1011 and 5 x 1011 vg per animal (i.e., 10 and 50 µL of vector at 1 x 1013 vg/mL titer... and 1 x 1012 vg*. Most systemic capsids are easy to produce in high titer, but higher doses (>1 x 1012...selecting a capsid, while Table 1 gives a brief overview of published descriptions of commonly used or emerging...before using it in an application.     Figure 1: AAV toolkit for targeted gene delivery to the brain...Capsids page to learn more about that collection. Table 1: Systemic capsid plasmids available from Addgene ...AAV9. At lower viral titers than AAV-PHP.B (e.g., 1 x 1011 vg/mouse), AAV-PHP.eB can transduce a greater...Diverse Brain Cells. Annual Review of Neuroscience, 45(1), 447–469. https://doi.org/10.1146/annurev-neuro-111020...

...plasmids. The Zhang lab, on the other hand, uses a 1 plasmid, single infection system. Genome wide mouse...X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F. Science. 2013 Dec 12. (PubMed...Wang’s library is designed to target a smaller set of genes and it includes more guides per gene. The Sabatini...Two new human lentiviral CRISPR library systems described in companion papers in Science in December were...Additionally, a concurrent Nature Biotechnology article describes the development of a mouse lentiviral CRISPR library...

... to other technologies like CRISPR. Figure 1: An overview of the general schematic of CRISPR/Cas... can be detected – in the zeptomolar range Time: ~1 hour Equipment required: 37°C water bath and 42°C ...nearly single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification... Not mentioned in the paper, likely approximately 1-2 hours Equipment needed: Mini PCR machines by DIY-Bio...Sensitivity: – 110 femtomolar  Time: Not mentioned, likely ~1-2 hours Equipment needed: PCR machines/Heat blocks...flattening the curve, it has become evident that widespread testing is crucial in controlling this pandemic...one temperature. Two Cas12a-crRNA complexes are designed, each binding to one strand of the reverse-transcribed...

...well, so let’s go over the details step-by-step. 1. Digestion Set up restriction digests for your insert...recipient plasmid : insert ratio” of approximately 1:3. Since the number of base pairs for each varies,...cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent cells...at minimum, two transformations after a ligation: 1. Control Transformation containing the ligation mixture...result in few colonies on both plates (unsuccessful 1), in a vector alone plate with many more colonies ...single enzyme if your insert is flanked on both sides by that enzyme’s restriction sites, but the insert...the orientation of your insert. You may want to design a diagnostic digest for this purpose. Ideally, ...