...DNA and processed the mixed sample in our pipeline (see Figure 2, sample 2). When we blindly analyzed ...look for potential contaminants. The analysis is a 2-step process using Geneious software: First, the individual... mean the sample is contaminated? No - what really matters is the number of hits to the same sequence ...these known impurities, as was the case for clean sample AAV-44362. In addition to these expected hits, ...sequence (gene). In a clean sample, there are typically fewer than 10 hits to a given sequence, and we believe... the contaminant we mixed into the original AAV sample at 5%. Detecting 5% contamination is a good start...low we could go. We prepared a range of spiked samples from 0.1-20% and discovered that, with a high enough...

...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...recombination so that the transgenes can be inherited on the same chromosome. Targeted Insertion allows for direct...variants of YFG because the transgenes are found in the same locus across fly lines (i.e. there won’t be positioning...

...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),...Retrograde Access to Projection Neurons. Neuron, 92(2), 372–382. https://doi.org/10.1016/j.neuron.2016.09.021...diabetes.55.04.06.db05-0927 Wu, Z., Asokan, A., & Samulski, R. J. (2006). Adeno-associated virus serotypes....2006.05.009 Wu, Z., Miller, E., Agbandje-McKenna, M., & Samulski, R. J. (2006). Α2,3 and α2,6 N-Linked Sialic Acids...

...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...overall inflation of USD has gone up 19.8% in the same time period. For ease of calculations*, let’s split...inflation at… roughly 34.9%, which means that the same plasmid would cost $155.25 in reagents and consumables...

...by a factor of 2. This means that if you set up two identical assays with the same sample, you could end...amplitude measured for each droplet in each well (Figure 2). A clean ddPCR should have a clear separation between...as low as 1C/s. Increasing the elongation time to 2 minutes and the denaturation time to 1 minute has ...standard curve is giving an accurate readout of your samples.  Of course, if qPCR were perfect there would be...must serially dilute your virus before adding your sample to a mastermix. At Addgene, we typically load 3... a wide range of titers. We usually dilute our samples 1:6 million to 1:25 million.  After making the ...and droplet oil that you use should come from the same manufacturer. Otherwise you will end up with poor...

...or non-homologous end joining (see Figure 2).  Figure 2: Schematic representation of integration...due to insertional mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you ...vectors: progress and applications. Gene Therapy, 17(2), 150–157. https://doi.org/10.1038/gt.2009.135. PubMed...efficiently during infection. Journal of Virology, 70(2), 721–728. https://doi.org/10.1128/JVI.70.2.721-728.1996...performing quantitative PCR titration of vector DNA in samples harvested 24 hours post-transduction (Wanisch and...

...the absence of genome editing (Fig. 2 and 3) (5, 3). Figure 2: Gene activation with nuclease-competent...shown to exhibit more limited activity than SpCas9 (2). In many cases this is due both to lower relative...PMID: 24076762. PubMed Central PMCID: PMC3844869. 2. Chari, Raj, et al. "Unraveling CRISPR-Cas9 genome...2015): 823-826. PubMed PMID: 26167643. 3. Kiani, Samira, et al. "Cas9 gRNA engineering for genome editing...

...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...assessment of many protein targets in a single sample; 3) producing recombinant, partially humanized rabbit...

...the result in Figure 1 could look like this: Figure 2: Expression of Plasmid B (as internal control) This...down expression of Gene X but, as shown in Figure 2, this was likely due to the original transfection ...PMID: 20074371. PubMed Central PMCID: PMC2817684. 2. Replicates and repeats—what is the difference and...were derived from the same source and were incubated on the same plate at the same time. Although the replicate...separate wells (within the same plate) with purified plasmid from the same aliquot/preparation. The replicate...provide the baseline/standard against which other samples can be compared. The Empty Vector Control (mentioned...control in this experiment could be an shRNA—in the same backbone as Plasmid A—that does not target any mammalian...

...CcdB.” Biotechniques. 1996 Aug;21(2):320-3. PubMed PMID: 8862819. 2. Bahassi, EM., et al. “F plasmid CcdB... desired insert should not grow.   Figure 2: Image from http://teachline.ls.huji.ac.il/72682/tutorials... the ccdB aspect, Gateway takes advantage of the same principle that cells will not propagate while expressing.... While these two strains ultimately perform the same function, there is some evidence that ccdB Survival...

...indicating primer dimers. (c) A double peak indicating 2 amplicons. Image credits (respectively): Zuzanna K...details for reproducibility’s sake!   Fig. 2: Setting up triplicates in multiple configurations...used method is the Standard Curve method. See Table 2, (adapted from Wong, 2018) for a comparison of different...Statistically defined log-linear phase   Table 2: A comparison of different qPCR analysis methods. ... for RNA quantitation. Biotechniques. 2000 Aug;29(2):332-7. doi: 10.2144/00292rv02. PMID: 10948434.Taylor...individual values match across the board. If the samples or sample conditions change, the multiplexed protocol...validation data, be aware that different machines, samples, and treatments may affect the assay. It’s unusual...

...from the same species and 2) these fragments will be more likely to bind (and block) the same epitopes...epitopes recognized by the secondary.   Figure 2: There are a few common solutions to avoid background...only antibody option is from the same species as your tissue samples. The bad news: this situation is ...excess secondary with serum from the same species as your primary/sample, and then applying this mixture to...processing and sample preparation can also help reduce the levels of endogenous IgGs in your samples, but know...The culprits are endogenous IgGs in your tissue sample. Most common IHC protocols use two antibodies -... the first antibody. When endogenous IgGs of the same species as your primary antibody are present, your...

...the system outlined in Schmid-Burgk, J.L., et al (2). In this post, I’ll cover two great applications ...molecularly evolved from mNeptune2 through 33 mutations and 2 deletions. CyOFP1 has a higher quantum yield, brightness...luciferin 24 hours later demonstrated a more than 2-fold brighter signal from Antares as compared to Fluc...PMID: 22894855. PubMed Central PMCID: PMC3501149. 2. Schmid, J.L., et al. (2016) CRISPaint allows modular...NLuc requires 100-fold less protein to produce the same amount of light from an Fluc or RLuc reaction. In...protein that could be co-imaged with GFP using the same wavelength for excitation. The result was the cyan-excitable... K., et al. (5) imaging multiple proteins at the same time in live cells using fluorescent proteins (FPs...

...counterstaining, mounting sample on slides, and confocal   Figure 2: High-level overview of an...into the several steps outlined below and in Figure 2. For a deeper dive into the steps and techniques, ...recognize the species of your primary antibody and 2) is conjugated to a fluorophore that suits your experiment...multiple proteins relate to one another in the same sample. For this strategy to work, it is important to...you use will depend on the target and sample type. Sample Preparation This step can involve multiple...target and sample type. Primary Antibody This is the first antibody applied to your sample, which will...of cells found in my tissue.” It is based on the same principles as other antibody-based assays, like ...

...the light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 ...inactive, dead Cas9 protein (dCas9) fused to CIB1; and 2) the activator - the CRY2 photolyase homology region... fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of full-length CRY2 and the...researchers saw an 11-fold increase in mRNA levels after 2 hr and a 400-fold increase after 30 hr. The system...authors fragmented Cas9 into N-terminal (residues 2-713, N713) and C-terminal (residues 714-1368, C714...nMagC714 instead of nMagHigh1-C714, generating paCas9-2. This change did not significantly alter the system...prior work, the Sato lab also showed that the paCas9-2 system could be spatially controlled and reversibly...

...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...possible, use low-bind or siliconized tubes to store samples as it is more difficult for viral particles to ...

...neoblast cells and the gene Prohormone convertase 2 (pc2) labels the central nervous system. Evaluating... characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching a specific mRNA sequence...223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver...Students can produce DNA templates via PCR off of the same plasmid and use these to create riboprobes for in...WISH protocol involves preparation of the tissue sample, several washes, and temperature changes to visualize...

...our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer team...columns are shown in red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down...Filling out and sending the deposit spreadsheet Figure 2: Example of the deposit spreadsheet showing the first...contact Addgene. We may require that you send your samples to Addgene pre-transformed in this strain. ...with instructions on how to prepare and ship the samples to Addgene and contact your technology transfer...

...to plasmid loss (Diaz et al., 2015).  Figure 2: The partitioning problem. For low copy plasmids...of plasmid into a separate daughter cells (Figure 2) (Ebersbach et al., 2005). However, more recent studies...Plasmids. Microbiology and Molecular Biology Reviews 62(2): 434–464. Pubmed PMID: 9618448 Diaz R, Rech J, Bouet... Plasmids with the same replicon are incompatible because they compete for the same replication control...incompatibility due to having the same replication machinery. High copy plasmids with the same centromere-like binding...if they have the same centromere-like region, and the NTPases end up “walking” the same plasmids to one... when multiple plasmids within one cell have the same replicon and/or partitioning system. Let’s start...

...base pair to another base pair at a specific site. 2. Temporal, spatial, or conditional control of CRISPR...difficult-to-engineer Sulfolobus islandicus rod-shaped virus 2 (Mayo-Muñoz et al., 2018). Only viral particles that...Editing of Sulfolobus islandicus Rod-Shaped Virus 2. Viruses 10:695 . https://doi.org/10.3390/v10120695...reducing editing from ~30% in control samples to 0-10% in Acr samples. Acr proteins can also be used to prevent...