...reactions. (Need a refresher? The five rules are: (1) don’t use the same overhang twice; (2) avoid palindromes...Gate) pot (Pryor et al., 2022).     Figure 1: Workflow for high complexity Golden Gate Assemblies...different enzymes and cycling conditions (see Table 1).   Enzyme Temperature Incubation time/cycling...cycling Overhang length T4 DNA ligase 25 °C 1 hour 3-base or 4-base T4 DNA ligase 25 °C ...hours 3-base or 4-base T4 DNA ligase 37 °C 1 hour 3-base or 4-base T4 DNA ligase 37 °C ...Ligase MM 37-16 °C cycling 3-base Table 1: Enzyme and cycling conditions available in DAD tools...assemblies are possible, the two sets of data together indicate that 35-pieces is a reasonable upper limit for...

... 5’ and 3’ homology arm repair template (Fig. 1, Step 1). There is no need for PCR or cloning, as digestion...selectable marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type ...correctly assembles the final targeting vector (Fig. 1, Step 4), and co-injection of the targeting vector...human cells. To use CRISPaint, users need 3 vectors:  1) a gRNA vector that targets the gene of interest, ... Tagging with Short Homology Donors.” Cell Rep.15(1) (2016):210-8. PubMed PMID: 27052166. Kunzelmann,...Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans.” Genetics. 202(4)...

...coding sequence in the antisense orientation (Figure 1, top). The entire DNA cassette would be flanked by...include both Cre and FLP recombinases.   Figure 1 (modified from Schnutgen et al. Nat Biotech 2003):...identical sites on one end of the DNA fragment (Figure 1, middle). A second recombination event then excises...between the identical loxP or lox511 sites (Figure 1, bottom). Since only one loxP and lox511 site will...genOway press release for more information. References: 1. Schnütgen F, Doerflinger N, Calléja C, Wendling O...sites directs the Cre recombinase to invert, translocate, or excise a DNA fragment. The availability of... the function of various point mutations and truncations in the absence of the endogenous gene. Spatio-temporal...

...experiment.     Table 1: Most popular guides, protocols, and collections of 2023. Educational Resource Top...visitors in 218 countries. In Table 1, you can see our most popular educational resources for each type of resource...our repository to an amazing 111 countries (Figure 1)! But while we were making the materials available...accessibility varied country to country.   Figure 1: Number of countries Addgene distributes to per year...date, we have partnered with six distributors (Table 1), in China, Korea, Japan, Brazil, India, and Mexico... execute a step can provide needed clarity (Table 1). Our most popular video, however, is “What is a Plasmid...logistical barriers, allowing people to access a wide catalog of plasmids from all over the world. This idea,...

...infection, gene targeting vectors with relatively short (1 kb) homology arms underwent homologous recombination...sequences of interest to cells with efficiencies of 1-40% after G418 selection (Kohli et al 2004, Kim et...with polylinkers for the addition of gene-specific 1 kb homology arms flanking a FLOXed neoR gene. pAAV-SEPT-Acceptor...since it routinely generates knockin efficiencies of 1-40% with G418-selected clones. However, there are ...efficiency of gene editing is occasionally less than 1%, and virtually never exceeds 40%. While this is comparable...gene inactivation in human cancer.     References 1. Russell, David W., and Roll K. Hirata. "Human gene...gene at once. Therefore, applications that require simultaneous modification of all alleles of a gene,...

...Correct Path.,” J. stem cell Regen. Biol., vol. 1, no. 1, Jan. PubMed PMID: 26702451. PubMed Central PMCID...known upper limit [1]. The piggyBac™ technology can be used for numerous applications, including transgenesis... Figure 1: Mechanism of the piggyBac™  transposase/transposon gene modification system and the ...genetic manipulation in mammalian cells until 2005 [1]. Like other transposons, piggyBac™ has two components...The predicted frequency of ‘TTAA’ in the genome is 1 in every 256 base-pairs of DNA sequence, making it...technologies and strategies for their projects. References 1. L. E. Woodard and M. H. Wilson, “piggyBac-ing models...technology that may be used for a variety of research applications, including making transgenic cell lines and ...

..., rather than a broadly  reactive secondary (Fig. 1). For example, an anti-mouse IgG2a secondary would...IgG2a>IgG2b>IgG1 (Manning 2012).      Figure 1: Broadly reactive versus subclass-specific antibodies...antibody binding and therefore the signal strength (Fig. 1). Monoclonal primaries If you do decide on a monoclonal...raised in a different species.      Table 1: Isotype considerations when choosing antibodies ...Elsevier. https://doi.org/10.1016/s0091-679x(08)60255-1  Manning, C. F., Bundros, A. M., & Trimmer, J. S. ...your experiment (all well validated for your application, of course!) When the stars are aligned and the...factors that affect isotype choice. Target and application should be considered The first question you may...

...For these experiments, cells were infected with a 1:1 ratio of two AAVs,  one expressing Cpf1 and the other...comparison of Cpf1 vs. Cas9, see the table below. Table 1. Comparing the Cas9 and Cpf1 CRISPR Nucleases   ...Overall, these approaches have two main drawbacks: 1) Most rely on transfection of more than one vector...leave them in the comments below.        References 1. Zetsche et al. "Multiplex Gene Editing by CRISPR–... a simple and effective method with multiple applications (in vitro, in vivo, transfection, viral transduction...Resources Find CRISPR Plasmids for Your Research Catch up on Your CRISPR Background with Our Guide Pages...

... microscopy contrast technique in modern biology (1). When performed correctly, fluorescence microscopy...relatively thicker three dimensional sample (see Figure 1); similar to an MRI or CT scanner. This is accomplished... the resolution of a light microscope (see Figure 1). What microscopy technique should I use for my experiment...new sample I always start by asking two questions: 1) Is the sample dynamic or static? 2) Is the sample...modern day fluorescence microscopes (see Table 1). Thin dynamic samples Ex: monolayers of live cells with...excitation light can push imaging depths closer to 1 mm. Traditionally, 2-photon has been a very slow imaging...manufacturers have similar information References 1. Lichtman JW, Conchello JA. Fluorescence microscopy...

...research experiences to undergraduate students (1, 2), but educators face significant challenges in designing...6). Methionine synthesis in S. cerevisiae (Figure 1) occurs through a well-characterized pathway with ...sequences for ten different MET/Met genes (Figure 1) from the two yeast species into the pYES2.1/V5-His...selected from different parts of the Met pathway (Fig. 1). In addition to the complementation experiments that...about Clare at the Biology Dept. site.  References 1. American Association for the Advancement of Science... become very different due to a whole genome duplication and extensive rearrangements in the S. cerevisiae...overexpression vector. This plasmid contains origins of replication and selectable markers that allow students to...

...type of assay is a clonal cell line known to contain 1 integrated copy of the qPCR target gene. Developing... successful transduction experiment. References 1. Kutner, Robert H., Xian-Yang Zhang, and Jakob Reiser...Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors."Nature protocols 4.4 (2009... titration methods."BMC biotechnology 6.1 (2006): 1. PubMed PMID: 16836756. PubMed Central PMCID: PMC1534021...events." Genetic vaccines and therapy 2.1 (2004): 1. PubMed PMID: 15291957. PubMed Central PMCID: PMC514534...particle. Consequently, titers based off of p24 quantification tend to be overestimates since they can include...quantify the amount of PCR product that comes from amplification of the original plasmid which can over-inflate...

...but also others including pseudorabies virus type 1, vesicular stomatitis virus (VSV; Beier et al., 2011...Skeletal Muscle AAV1, AAV6, AAV7, AAV8, AAV9 Table 1: Common AAV Serotypes and their target tissues (adapted... serotypes capable of entering neurons (see table 1)(Choi et al., 2005; Taymans et al., 2007; Howard et...efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310...Wolfe, J.H. (2014). Adeno-associated virus serotypes 1, 8 and 9 share conserved mechanisms for anterograde...,9 in rat neurons and glia in vitro. Virology 372(1), 24–34. PubMed PMID: 18035387. PubMed Central PMCID...transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem...

... Figure 1 below) was free to anneal to complementary ssDNA molecules in vitro. Figure 1: Cas9 binding...Corn Lab Members who worked on this publication:         References 1. Richardson, Christopher D., et al...allowed sequence replacement rates of approximately 1%. This is by no means a large number, but it was attained... reagent. This interplay falls into two broad categories and thus, causes two types of editing outcomes...altering its function). HDR is crucial for certain applications, for example, correcting the allele that causes... dissociation rates and discovered that the catalytically active form of the nuclease dissociates very...we measured identical dissociation rates for catalytically inactive dCas9 molecules. We followed these ...

...(a) (b)  (c)   Fig. 1: Melt curves showsing (a) a single peak, indicating one amplicon and no primer...PCR products in a rich web application, Bioinformatics, Volume 27, Issue 7, 1 April 2011, Pages 1019–1020...relatively easy to multiplex (Thellwell, 2000). See Table 1 for a comparison of features of several commonly used...in primer Yes Yes Agarose gel Table 1: Features of various qPCR chemistries. Adapted from...changes in ΔΔCT are represented by RQs between 0 and 1. Flipping your experimental and control values during...see this fold change as a positive difference (RQ>1), which may be easier to conceptualize. Just remember... in Molecular Biology, vol. 1160, DOI 10.1007/978-1-4939-0733-5_3, © Springer Science+Business Media New...

...building it! Table 1 has a list of Fragmid destination vectors currently available. Table 1: Fragmid destination...components on the Fragmid website.   Figure 1: (Top) Schematic overview and timeline of the Golden... CRISPR vector the Fragmid way!    Figure 1: Selecting your vector type on the Fragmid website...components available will depend on your answers in step 1, so you’ll only be able to select components that ...ampicillin resistance cassette and an origin of replication, which are not schematized. Sizes of fragments...five months. When they tested those clones (in duplicate), 93% had the correct banding pattern in the restriction...correct. Because they created the assemblies in duplicate, they were able to confirm that at least one plasmid...

...not let the panelists introduce themselves. I do a 1-2 minute introduction. If you chose good panelists...take 5 minutes to introduce themselves and, voila, 1/2 of your time is gone with no interesting discussion...introductions. The moderator asks questions, calling on 1-2 panelist for each question in a way that elicits... time you are at a talk and the speaker goes over 1 hour, watch what happens to the attention of the audience...postures shift, they are checking out. If you can, plant 1-2 good questions with trusted audience members. A ... about making sure the audience is interested, educated and talking as a result of the content. Here are...contribute. The moderator should identify the funny, provocative or interesting stories that will make for a fun...

...mechanical restraint Location: back or hind legs Gauge: 25–27 Volume: < 2–3 ml (~1 ml per site) Skill required...! Table 1: Summary of common injection routes in mice. Injection Route Restraint Location Gauge Volume... advanced and finally, to expert.   Figure 1. Visual representation of common injection routes ...hind legs; between skin and muscle 25–27 < 2–3 mL (~1 mL per site) Beginner Intradermal Anesthesia Between...resources on the Addgene blog Mouse Modeling Part 1 Mouse Modeling Part 2 Five Popular Model Organisms.... It sounds complicated but has a pretty low learning curve! Restraint: scruffing Location: throat Gauge... Restraint: mechanical restraint or anesthesia Location: tail vein or retro-orbitally Gauge: 27–30 Volume...

...Cambridge office – previously at 1 Kendall Square, but newly relocated to 75 Sidney Street. Then the plasmids...here at Addgene – 500,000 plasmids shipped! That's a 1/2 million plasmids sent to happy scientists around...Have a question? Email: [email protected] Call: +1 (617) 225-9000         ... thank you. 500,000x Thank You! Addgene's new location: 75 Sidney St, Suite 550A, Cambridge, MA 02139...

...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.,...

...disadvantages: Table 1 (Joglekar et al., 2017)  Table 1 (Gutierrez-Guerrero et al., 2020) Table 1 (Cronin et al...polyA tail.   Figure 1: Lentiviral production uses three plasmids: (1) The transfer plasmid, (2)... specific purposes as described previously (Table 1, Gutierrez-Guerrero, et al, 2020). Because retrovirus...The glycoprotein will be incorporated into the replicating rabies virus envelope, travel retrogradely, and... Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene...