...reserve a small aliquot, split into two parts to (1) run a DNA gel to confirm fragment size and (2)  incubate...with a phenol-chloroform extraction.    Fig. 1: Schematic overview of ChIP. Image courtesy of Shengliu... let’s do a quick review. At this point you have (1) crosslinked your proteins of interest to any DNA ...Fragmenting  The next step is break the DNA into random, small pieces of roughly equal size, typically ...enzymatic digestion. Sonication provides a truly random digestion, but is harsher and requires more optimization...your protein of interest is a low abundance transcription factor or cofactor. In such cases, the gentler...

...dual-color control of neurons. Nature Communications, 12(1), 1–20. https://doi.org/10.1038/s41467-021-24759-5 Zhang...to consider when choosing one.     Figure 1: Example of an experimental optogenetic setup. A light... 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_2 Lehtinen, K., Nokia, M. S., & Takala... 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_2 Boyden, E. S., Zhang, F., Bamberg, E... charged ions to rapidly pass through the cell membrane and initiate an action potential - a very useful...and close when exposed to light and change the membrane potential. OptoXRs are modified GPCRs. When exposed...downstream signaling pathways that can change both the membrane potential and a host of other cellular functions...

...control exactly in which locus your transgene ends up (1): Random insertion has the advantage that you...pound symbol”. References 1. J. R. Bateman, et al. Site-Specific Transformation of Drosophila via PhiC31... you’d like to use to generate your new fly line: 1. Where would you like the gene to be incorporated ...you need to combine multiple transgenes in one fly, having the transgenes in different loci allows for...use? Random insertions take longer because you need to determine in which chromosome your transgene has...brief introduction as to how you can engineer new transgenic flies to study your favorite gene (YFG).  Sometimes... is thrillingly new, you might have to make a transgenic fly in order to study it. The process is fairly...

... emailed submission) in the Deposit menu: Figure 1: Begin the deposit process by clicking "Submit Online...cases Please contact us at [email protected] or +1 (617)-ADDGENE (233-4363) if your deposit falls into...Addgene?" or contact us at [email protected] or +1 (617)-ADDGENE (233-4363) with additional questions...strain. Please contact us at [email protected] or +1 (617)-ADDGENE (233-4363) if you’d like to distribute...page so that any data you enter is not lost.   Step 1: Sequences, Maps, and Files Please provide any full...a 1.5 mL microfuge tube at a concentration of 0.1–1 µg/µL (or bacterial streaks, if you prefer). If you...number of unique plasmids in a pooled library can range from a few hundred to millions. Pooled libraries...

...indicates the presence of a multimer.   Fig. 1: The monomer of Plasmid 12259: pMD2.G (depicted on...plasmid sequencing services using long-read sequencing (1-25 kb), such as Oxford Nanopore sequencing or PacBio...10017-26. doi: 10.1074/jbc.M110.176636. Epub 2011 Feb 1. PMID: 21285356; PMCID: PMC3060453. Bazaral M, Helinski...multiple circular DNA forms of colicinogenic factor E-1 from Proteus mirabilis. Biochemistry. 1968 Oct;7(10...Methods Mol Biol. 2009;582:11-25. doi: 10.1007/978-1-60761-340-4_2. PMID: 19763938. Maucksch C, Bohla A...size, multimers may exhibit reduced transformation and transfection efficiency compared to the monomeric... Elfinger M, Hartl D, Rudolph C. Transgene expression of transfected supercoiled plasmid DNA concatemers...

... tissue. References 1. Aschauer DF, Kreuz S, Rumpel S. Analysis of transduction efficiency, tropism and... and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 2013 Sep 27;8...M. Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an...AAV5, AAV8) enable different patterns of transduction and transgene expression across different cell types...expressed from your cell line of choice after transduction with this virus. While various Cas9 mutants ...classic Cas9 nuclease, which introduces double stranded DNA breaks, can be expressed from lentiCas9-Blast...single stable Cas9-expressing cell line and then transduce this line with lentiviruses expressing various...

...unleashes its cargo – its own genome.   Fig. 1: Viral particle infection into a host cell. Image ...Vector Resources at Addgene! References and Resources 1. Roe T, et al. “Integration of murine leukemia virus...nuclear import of human immunodeficiency virus type 1 preintegration complexes.” Proc. Natl. Acad. Sci. ...one they have a high transduction efficiency and can effectively infect a wide range of cell types that ...used to achieve transient, high expression of the relevant cargo without the fear of random genomic integration... Packaging Capacity (>8kb) High Transduction Efficiency Transient Gene Expression High Immunogenecity...viral envelope, which fuses with the target cell’s membrane, allowing the viral particle access into the cell...

...-HA frankenbodies include mEGFP, mCherry, and HaloTag. Figure 1: (a) Anti-FLAG frankenbody (FB)...Comm. 2019. https://doi.org/10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a... to Cas9’s larger expression requirements (Figure 1), but Cas12a has generally been underutilized in genetic...Here's what you'll find in this post: Anti-FLAG Frankenbodies for modular imaging of cellular targets Optimized...mammalian genome regulation and editing Anti-FLAG Frankenbodies for modular imaging of cellular targets by: ...: Mike Lacy Adding to their set of anti-HA frankenbodies (Zhao et al. 2019), Tim Stasevich and Hiroshi... have recently published a set of anti-FLAG frankenbodies, a flexible, modular tool for labeling. Here...

...available from their original sources. Figure 1: Biological repositories that distribute mouse strains... from Reproducibility for Everyone. Case study 1: Cell lines Many of the reagents used to investigate...observational study. The Lancet Microbe 1:e14–e23 . https://doi.org/10.1016/s2666-5247(20)30004-5 McCray...scientific community. Addgene was awarded a Fast Grant to ensure that the scientific community will continue...-2. The resurgence in popularity of the hACE2 transgenic mouse strain, for example, is sustained by the...University of Iowa (McCray et al., 2006), the hACE2 transgenic mice were designed after it was discovered that...Deposits of new reagents, policies from journals and granting agencies requiring materials to be deposited into...

...s pLKO.1 protocol. Pro-tip: this plasmid grows more slowly than standard plasmids. Find pLKO.1 - TRC cloning...experiments.  Find the scrambled shRNA vector. pLKO.1 - TRC cloning vector - This vector from the David ... different cell types so it is useful in a wide range of applications. This plasmid was the top requested...pSpCas9(BB)). The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP (PX458). lentiCRISPR...lentiCRISPR v2 - This 3rd generation lentiviral transfer vector also from the Zhang lab is used to deliver...David Root lab is a 3rd generation lentiviral transfer plasmid for cloning and expressing new shRNA sequences...the necessary elements for packaging, reverse transcription, and integration needed for the production of...

... the samples stored in our lentivirus tubes (Fig. 1). However, once thawed, there was a substantial reduction...in the pH of samples stored in our AAV tubes (Fig. 1). It’s important to note that the change in pH only...using the thawed virus within 24 hours. References 1. Murphy BM, Swarts S, Mueller BM, van der Geer P, ... However, the pH changes in our solutions are transient and return to normal once the tube is opened and...restored. We have no evidence to suggest that the transient change in pH affects AAV function. Furthermore...Furthermore, another study tested AAV infectivity after a range of pH exposures. They showed that AAV infectivity...MC, Fitchmun MI. Protein instability following transport or storage on dry ice. Nat Methods. 2013 Apr;10...

...digital) signal at the end of the reaction (Figure 1). Partitions that did not contain any target DNA at...integrated through homology-directed repair (HDR) (1), or indel mutations resulting from non-homologous...Follow him on Twitter @AnotherLabRat.     References 1. Miyaoka, Y. et al. Isolation of single-base genome-edited...edit. Since exploitation of NHEJ to generate translational reading frame-altering indels for “functional... an unedited site distal tothe predicted double strand break, and a NHEJ/ “drop-off” probe designed to...to bind directly at the predicted double strand break site (Figure 2A). The single molecule resolution...PubMed PMID: 26914317. 4. Mock, U. et al. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates...

...flanked by ~50-150 bp transposon end sequences (R and L ends).  Figure 1: (A) Components involved...CRISPR-associated Transposase) by the Zhang group. Mechanism of CRISPR-transposons One of the CRISPR-transposon systems...DNA transposons have been hindered by their limited programmability: depending on the transposases involved...-guided transposition. By natively combining the DNA integration capabilities of transposases and the ...crRNA.  Three transposase proteins (TnsA, TnsB, TnsC). The donor DNA (aka mini-transposon), which contains... each type of CRISPR-Transposon system. (B) Mechanism of RNA-guided transposition in type I-F INTEGRATE.... Separately, TnsABC forms a transposase complex with the mini-transposon, excises it from the donor molecule...

...skeletal muscle contraction.   References 1. Degorce, François, et al. "HTRF: a technology tailored for..."Förster resonance energy transfer measurements of ryanodine receptor type 1 structure using a novel site-specific...standard for genetically-encoded FRET donors (Fig. 1). Scores of FP variants have been created, thereby...with finesse using tRNA suppression technology (Fig. 1) (Dumas, Lercher, Spicer, & Davis, 2015). By using...orthogonal labeling strategies are presented (Fig. 1) which can be carried out using Addgene plasmids. ...they cannot cross cell membranes, so they are best used either with plasma membrane proteins (such as GPCRs... minimum energy transfer values you can expect, thereby establishing the dynamic range of your FRET assay...

... experimented with a group mentoring format where 1 mentor met with a group of mentees to get more “bang...The groups in these programs typically consist of 1-2 mentors and 3-5 mentees who meet together for an...get together. If you all work at the same place, a 1-2 hour lunch on a regular schedule might work. If ...meeting at both the Fox Chase Cancer Center and Brandeis University. 5 easy steps to forming your own peer... of people who are all interested in making a transition. Schedule your first meeting and get organized... your successes and support each other as you transition to the next stage in your careers! Stay in touch...

...effective recovery of knock-ins, but did result in 1-2 kilobases of additional sequence being introduced...facilitating recovery of edited animals. Figure 1: In the background are 504 restriction digests of ...selection for repair of a temperature-sensitive pha-1 mutation significantly enriches for knock-in of 2x... 3xFLAG epitopes into other, non-linked loci; pha-1(e2123) mutant worms are perfectly viable at 15 ºC,... repair pathway. Reagents required to perform pha-1 co-conversion are available through Addgene. In 2019... technologies have transformed the palette of experiments possible in a wide range of organisms and cell...genomic locus (CRISPR-ChAP-MS) could further transform the range of experiments imaginable in C. elegans. ...

... levels. That is, if transcription factor 1 controls expression of transcription factor 2, which directly...by modulating the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information...difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away is suitable for...expression, Aranda-Díaz et al transformed yeast with both a GAL4/estradiol controlled transcription factor ... a broad pH range. The four biometric probes successfully targeted endosomes under a range of pH conditions...ligand binding domains and the MSN2 transactivation domain. Transcription factor activation via the appropriate...progesterone controlled transcription factor as well as reporters specific to each transcription factor. They then...

... help improve the transport of protein of interest the membrane.   Figure 1: Electron microscope...proportion were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic, because...expression of hM4Di in plasma membranes in both species (bottom panel in Fig. 1). Our results indicate that... the transgene of interest. In our studies, mCherry interfered with the transport to the membrane of the...the hM4Di might improve the transport of this DREADD to the plasma membrane. The haemagglutinin (HA) tag...the expression of the transgene of interest and its localization in the membrane. The lesson we learned... the plasma membrane. Note that the use of the HA tag significantly improves the transport of hM4Di to...

... Figure 1: Strand separation and target DNA binding to the Cas9 nuclease. Stable strand separation ...by 2 sets of interactions : 1. Interactions between the non-target strand and a positively charged groove... improvements in genome engineering! References 1. Slaymaker, Ian M., et al. "Rationally engineered ...increases when target DNA strand separation is stabilized (2,3). Stable strand separation is maintained...and therefore destabilize basal strand separation. This decreased strand separation combined with the weak...the non-target strand and the HNH/RuvC groove, and interactions between the target strand and the gRNA....nuclease domains 2. Interactions between the target strand and the gRNA (see figure below) Because off-target...

...Figure 1: The Addgene Requests Dashboard   The dashboard will display the following items (Fig. 1): ...depositor, but it can also be valuable data to share in grant proposals or to demonstrate impact in annual reports...