...alkaline lysis plasmid miniprep Starting material: 2 mL bacterial culture Product: plasmid DNA This kit-free...protocol: 1) it solubilizes both DNA and agarose, and 2) it helps DNA stick to the glass. After binding DNA... had a similar level of sensitivity. Figure 2: Steps to purify DNA using a cellulose dipstick. Image...Academy of Sciences of the United States of America, 76 2, 615-9 . https://doi.org/10.1073/pnas.76.2.615  Zhou...plasmid purification and DNA gel extraction method described in Kim and Morrison is nearly identical to that... Image: Kim and Morrison, 2009. This method provides DNA yield and quality similar to that obtained...

...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Wang,...(see maps section for images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre...Digital PCR Method. Human Gene Therapy Methods, 29(2), 96–103. https://doi.org/10.1089/hgtb.2017.198   ...protocols, titering methods, and quality control are described below. AAV Production AAV distributed by Addgene...) are generated from lentiviral preparations (described above) that are subject to a concentration step...

...non-specific proteins, and then elute YFP.  Option #2: Opto-Nanobodies (OptoNBs) purification You skip adding...blue lights. Which option do you choose?  Option #2 may sound like fiction, but light-controlled protein...experiments.  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced...purification is now a reality thanks to OptoNBs. Recently designed by the Toettcher Lab, OptoNBs are photoswitchable... put AsLOV2 because here it was less likely to destroy the nanobody’s function than if inserted into the...blue-light induced binding. The lab used this general design scheme to generate two more OptoNBs, one against...exciting because using light to control signaling provides greater spatiotemporal resolution than pharmacological...

...removal of H3K4me1/2 and H3K9me2 histone modifications. Like the TALE-LSD1 system described above, dCas9-LSD1...form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters and...oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer. Oncogene 34...approach for targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc.2016.9 Find plasmids...which uses light to regulate transcription, also includes light-regulated histone methyltransferases and...certainly not the only CRISPR-based technology designed to alter gene expression. Fusing dCas9 to a transcriptional...epigenetic modifiers? As with any experiment, your desired outcome will determine the tool that you should...

...immediate early gene expression. Genes Dev. 1988 Jun;2(6):718-29. doi: 10.1101/gad.2.6.718. PMID: 2843425...Targeted Gene Expression in Drosophila.” Genetics 186(2) (2010): 735-55. PubMed PMID: 20697123. PubMed Central...2022). https://doi.org/10.1038/s41598-022-07852-7 De Boer, Herman A., Lisa K. Comstock & Mark Vasser. ... in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197-214. PubMed PMID: 22129153. Additional...to the promoter and turning transcription OFF. Despite their bacterial origins, Tet systems function well...promoters can be used in the repressible manner described above, as well as the inducible manner detailed...30 years since the original Tet-Off system was described by Gossen and Bujard, both the promoter and transactivator...

...PubMed PMID: 27463678. 2. Green, Alexander A., et al. "Toehold switches: de-novo-designed regulators of gene...certain states of interest. For example, in Figure 2 below we present a two-input, five-state GRSM register...linking RSM inputs to endogenous gene expression (2). Our paper (1) explains how to arrange attB-attP...also be used to design primers to read states by qPCR or Sanger sequencing as described in the Material...hammerhead ribozyme parts are described in Nielsen et al (3) and Lou et al (4). Despite choosing proper register...machine design space is highly degenerate and often times there are multiple register designs that may...recognition sites from the register. If the RSM is designed in such a way that each possible order of inputs...

...to (1) run a DNA gel to confirm fragment size and (2)  incubate with RNAse and Proteinase K to purify the...proteins of interest to any DNA they were bound to (2) fragmented the DNA (3) used antibodies to specifically...adjustments, to get everything just right!  Fig. 2: Goldilocks is actually celebrating a successful ChIP...immunoprecipitation (ChIP) is an extremely useful technique that provides insight into protein:DNA interactions. ChIP works...up the DNA, or enzymatic digestion. Sonication provides a truly random digestion, but is harsher and requires...both tissue or cell type, amount of protein, and desired fragment length for your output assay. A longer...

... the two DNA strands and releasing topoisomerase (2).  These days commercially available TOPO kits provide...Taq polymerase using your favorite PCR protocol.  2. Set Up TOPO Cloning Reaction: Mix together the PCR...PubMed PMID: 1658796. PubMed Central PMCID: PMC52876. 2. Novel approach to molecular cloning and polynucleotide...needed for TOPO cloning: 1. Create Your PCR Product: Design standard primers (no need to add unique restriction...

...use 2 different secondaries for your antibody pair and visualize both targets in parallel (Fig. 2). For...successfully visualize both proteins.     Figure 2: Two primary antibodies with the same isotype can ...In the case above, you can’t costain with your desired antibodies because the pair belong to the same ...

...). A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons. Neuron, 92(2), 372...proteoglycan (HSPG)3 AAV receptor (not strictly required)2 Human fibroblast growth factor receptor 1 (FGFR1... AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated nomenclature...suggest these serotypes as a good place to start. Table 2: AAV tissue tropism for mice in vivo. To target... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PloS One, 8(9),... capsid of another. Formally, pseudotypes are described with two numbers separated by a slash: the first...through our in-stock viral prep service.  AAV2 Despite being isolated second, AAV2 is the best studied...

... analogs such as 2-fluoroadenine (2-FA), organisms with functional APT will convert 2-FA to cytotoxic ...cytotoxic nucleotides, resulting in death. In the presence of 2-FA, potent gene silencing of APT is required for...selection of plants undergoing RNAi, and LIIbeta F 1-2 RNAi, which allows assembly of intron-spliced hpRNAs...eukaryotic process where approximately 20-30 nucleotides of double-stranded RNA (dsRNA) results in downregulation... of an RNAi-inducing transgene. Typically, the desired target of RNAi is expressed as inverted repeats... and quantify actively silencing plants. Non-destructive methods, most popularly with a fluorescent reporter...Fluorescent reporter-based RNAi constructs have been designed and widely implemented in plants, such as in the...

... 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010 167  Nature 3.3%  10.5%  3.2  534.1 3...Microbiology  1.6% 1.2%  0.75   125.3 In figure 2, you can see the full impact from using these weighted...plasmids deposited at Addgene still accrue on average 2 to 3 times as many citations as other papers in the... Science. For each publication, Web of Science provides basic bibliographic information and uses its vast...citations due to the journal of publication. Table 1 provides an example calculation of the weights. Table 1...

...drastically with lower titers. As you can see in figure 2, the expression from the 5x1011 titer was essentially... roughly 6-fold increase in expression [3, Figure 2]. Note how expression increases exponentially with...international 61.1 (2002): S9-S15. PubMed PMID: 11841606. 2. Schultz BR, Chamberlain JS. Recombinant Adeno-associated...if you end up concentrating your AAV above your desired titer and want to dilute it, err on the side of...less buffer than you would to achieve the exact desired concentration to avoid over-diluting, and make ...

... be directly used in the research on the SARS-CoV-2. Nine of the overall plasmid selection will be open... cloning grade DNA beta project We will ship you  2 µg of DNA in 20 µl of Tris buffer suitable for molecular...Plasmids 101 blog series Learn about the different grades of DNA  Read about the various ways to quantify...

...human transcription factors such as PAX7, PAX6, AP-2 alpha and ISLET-1. In addition, we distribute monoclonal... example through the use of less toxic adjuvants; 2) generating complex antibody chips that allow assessment... which results in neutralization or targeted destruction.  Antibodies are composed of two light chains...

...correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers that overlap at least...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...colonies (5-10) and grow them in a small culture volume (2 or 3 ml) containing the corresponding antibiotic, ...coveted plasmid with no substantial delays. Design, design, design Before diving into the experimental work...experimental work. Fragments generation After you’ve designed your cloning scheme, you’re now ready to generate...from a large genome, you can recheck your primer design or optimize your primers or PCR conditions. Purifying...second and more detailed look at your experimental design. Full lawn of cells. The antibiotic in your plate...

...lab found that H2B-GFP expressing cells exhibited a 2-4 fold increase in the number of endogenous indel ...Denham lab made several other small changes creating 2 other versions but these did not surpass GEmCherry2...fluorescence and in-frame fluorescence.  Figure 2: The GEmCherry reporter. Image from Højland Knudsen...created SRIRACCHA in 2017 to enrich and identify desired CRISPR mutants (Wen et al., 2018). SRIRACCHA uses...constructs for the reporter to work and to make the desired genome edit:  A donor plasmid that contains a ...OH-T and doxycycline. A gRNA that targets your desired gene (whose sequence is also found in the reporter...well. The ratio of RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  By using...

...Nature Cell Biology, 26(2), 294–304. https://doi.org/10.1038/s41556-023-01342-2 Koblan, L. W., Arbab, M...order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent...region of separate strands called an R-loop (Figure 2). This allows the guide RNA to base pair with the ...accessible on the outside of the enzyme. Figure 2: CRISPR R-loop. Created with BioRender.com. Base...), 35–40. https://doi.org/10.1038/s41587-020-0592-2 Additional Resources on the Addgene blog CRISPR 101...in the development of CBEs and ABEs. This post includes a lot of acronyms, so we’ve included a glossary... in different cell types, available at www.crisprbehive.design. CGBEs fused to pioneer factors to improve...

...one of lox2272 sites will now be sandwiched between 2 loxp sites and is excised by Cre (Figure 1). This ...flank the transgenes (non-homologous pairs) (Figure 2.). They found that almost all off-target expression...exercise caution when using them.    Figure 2. Schematic diagram showing the mechanism of spontaneous... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLoS ONE 8:e76310... Pseudotyped with Viral Capsids from Serotypes 1, 2, and 5 Display Differential Efficiency and Cell Tropism...experiments to minimize it. Overview of DIO/FLEx vector design Recombinase-dependent vectors utilize pairs of ...between them. Early recombinase-dependent vector designs used a lox-STOP-lox (LSL) or frt-STOP-frt (FSF)...

... will repeatedly nose-poke at the active hole [1, 2].    Place preference/place aversion also models reinforcement...spend most of its times in the opposite chamber) [2]. Laser activation is usually controlled automatically...two different ways 1) your hypothesis is wrong or 2) there is some problem with the technical implementation...PMID: 21716290. Pubmed Central PMCID: PMC3775282. 2. McDevitt, Ross A., et al. "Serotonergic versus nonserotonergic...other words, your optogenetic experiment should be designed around a behavioral test in which your circuit...of mouse transgenics. Everything that has been described is intended for the mouse; however, all the steps....Diester, Ilka, et al. "An optogenetic toolbox designed for primates." Nature neuroscience 14.3 (2011)...

...are many docking sites available for integration. 2. How soon would you like your flies ready for use?...beautiful immunofluorescent images like in this paper (2). Metabolic assays such as Seahorse can be employed...PMID: 16547094. PubMed Central PMCID: PMC1526508. 2. H. Jiang,  et al. Cytokine/Jak/Stat signaling mediates...Harvard University. In my previous two posts, I’ve described the fundamentals of how to work with Drosophila...Drosophila as an experimental model organism. I then described the Gal4/UAS system used by geneticists to study....e. there won’t be positioning effects). When designing experiments where you want to express multiple...wondering, “How do I know which of my flies have the desired integration?” Not to worry! In flies, phenotypic...

...required to make a plasmid. In 2023 (and at closer to 2 million plasmids shared than not!), we think it’s ...time does it take to make a plasmid? Figure 2: Survey results from a Twitter poll run in 2023, n...go wrong, replace them for free. Plasmids ship out 2-3 days after ordering, making it a toss-up as to whether...in science – the time spent by the scientists designing and performing the experiments. This cost varies...quickly with the postdoc who’s really good at primer design – but first, you have to know what resources exist...well) plate! How much do repositories help?   Designing precise calculations for any of the numbers in...engagement with and use of repositories like Addgene provides large-scale savings. How large? Using Doench’s...

...termed fluorescence activated cell sorting or FACS (2). By analyzing single cells instead of the population...expression. A routine flow cytometry experiment uses 2 or 3 different fluorescent colors each measuring a...distinct populations on a FSC versus SSC plot (Figure 2).  A user can then “gate” around the specific population...cell and T cell into distinct populations (Figure 2). An immunologist who studies B cells, might then ... B cells, plasma cells or others.   Figure 2: Due to differences in their size and complexity, ...assessed for the desired parameter. A droplet containing a cell that is positive for a desired parameter is...their observations. However, while flow cytometry provides analysis at the cellular level, it cannot be used...

...Central PMCID: PMC4151723. 2. Doench, John G., et al. "Optimized sgRNA design to maximize activity and ...CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the pAWp12... before beginning your experiments. Figure 2: Strategy for assembling barcoded combinatorial gRNA... Blog Learn Best Practices for Designing Your gRNA Read How Barcodes Can Be Used in Lineage Tracing Read...genetic perturbations. The ease with which one can design and synthesize CRISPR guide RNAs (gRNAs) for genome...their functions. The continual advancements in gRNA design necessary to achieve maximal on-target and minimal...regulation in biological systems. The CombiGEM platform provides a means to create barcoded gRNA libraries that...

...Step 2: Develop a research plan Most grant applications restrict the space available to describe the ...require application submission in-house approximately 2 weeks before the RFA due date in order for the administrative...proposal typically lists the deliverables, explains 1-2 potential pitfalls of the intended protocol, and offers...application. It typically includes overview information, key dates, funding description, research plan, award... setbacks, and potential alternative routes, Provides opportunities to convince others why a project...aspects are understood. While reading the funding description, pay attention to the keywords used and think...literature and offer educated insight into what the desirable features of future technologies should offer in...

...of other Cas proteins found in the so-called class 2 CRISPR-Cas systems. There are 24 variants of the cas14...the presence of infectious organisms (ex: SARS-CoV-2) and genetic mutations. By providing a gRNA specific... cas1, cas2, cas4, and a new gene cas14. cas14 encodes for a small Cas protein (40-70 kDa), which is half...Harrington, Lucas B., et al. "Programmed DNA destruction by miniature CRISPR-Cas14 enzymes." Science 362.6416...

...CcdB.” Biotechniques. 1996 Aug;21(2):320-3. PubMed PMID: 8862819. 2. Bahassi, EM., et al. “F plasmid CcdB...those that do not contain the desired insert should not grow.   Figure 2: Image from http://teachline.ls.huji.ac.il... plasmid maintenance during cell division. ccdB codes for the toxic protein (CcdB) that acts as a DNA ...death. ccdA, another gene found in the ccd operon, codes for the antitoxin protein (CcdA) that protects the...of ccdB, leading to positive identification of desired clones.   ...Becomes a powerful cloning tool...

... (2,3). Stable strand separation is maintained by 2 sets of interactions : 1. Interactions between the...formed by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand and the gRNA... Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal structure of ... nuclease activity of the Cas9 nuclease itself. Design and testing of eSpCas9 Looking at the structure...and the negatively charged DNA and therefore destabilize basal strand separation. This decreased strand... choice and get improved on-target specificity. Design and testing of SpCas9-HF1 Kleinstiver et al and...Joining Repairs Double Strand Breaks Get Tips on Designing Your gRNA Browse Our CRISPR 101 Series Resources...

...phosphorylation events in the proteomes of higher mammals (2). How can we begin to understand the importance of... the open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...optimized Sep-OTSλ and can recommend its use (6).  2. C321, a specialized bacterial host strain for pSer-incorporation...biology4.5 (2002): E127-E130. PubMed PMID: 11988757. 2. Hornbeck, Peter V., et al. "PhosphoSitePlus, 2014...site-specific incorporation of phosphoserine (as described below) provides researchers with a valuable tool for understanding...organisms lacked a classic cysteinyl tRNA synthetase, despite abundant incorporation of cysteine in their proteomes...pSer-incorporated proteins on a single plasmid. This includes 4 copies of a tRNA that specifically recognizes...

...preventing design of antibodies specific to each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained...Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow 3.2. ...M-CFP expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate...processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...R26R-Confetti described in Hugo J. Snippert, et al. (2010) and the MAGIC Marker strategy described in Karine...Brainbow-1 system employs Cre-mediated deletion, the design of Brainbow-2.1 cleverly combines Cre-mediated ...immediately following the promoter. The STOP cassette includes a mutant YFP that does not fluoresce, but can ...