...the mixed sample in our pipeline (see Figure 2, sample 2). When we blindly analyzed AAV-68544, we were...look for potential contaminants. The analysis is a 2-step process using Geneious software: First, the individual...Léger A (2015) Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus...especially important when producing multiple vectors in parallel because the risk of accidental errors increases... be used to identify DNA contaminants in AAV preparations and provide information on these DNA species...pioneering the systematic use of this new QC assay to guarantee that you receive the best AAV vectors for your...Identity of Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts. Human Gene Therapy ...

...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227... Find plasmids from this publication at Addgene. 2. Kleinstiver, Benjamin P., et al. "Genome-wide specificities...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674...also called Cas12a. In 2015, Feng Zhang’s lab characterized two Cpf1 nucleases, distant cousins of well-...Until recently, two major elements of Cpf1 characterization were missing: on-target and off-target editing...efficiency. Kim et al. and Kleinstiver et al. characterized genome-wide editing efficiency of two Cpf1 orthologs...lower than that of the widely used SpCas9 and comparable to SaCas9. As seen with Cas9 orthologs, Cpf1 ...

... fluorophore excitation by absorption of a photon 2. Energy transfer from the excited donor directly to...PMID: 24194740. PubMed Central PMCID: PMC3810607. 2. Bajar, Bryce T., et al. "A Guide to Fluorescent Protein...PubMed PMID: 10500161. PubMed Central PMCID: PMC18018. 2. Miyawaki, Atsushi, et al. "Fluorescent indicators...various papers to make the decision easier. 1. Müller, Sara M., et al. "Quantification of Förster resonance ...donor and acceptor fluorophores are fused to two separate proteins. (In contrast, intramolecular FRET probes...error in suboptimal or undetectable FRET, well-characterized FRET reference standards can be used to validate...

...procedure to be performed using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As...Primer design Using specific primer design (Figure 2), IVA cloning can be used to perform any plasmid modification... for each type of modification is shown in Figure 2, and is discussed in more detail below. The main requirement...Biotechniques 10.1 (1991): 62-66. PubMed PMID: 2003926. 2. Bubeck, Peter, Monica Winkler, and Wilfried Bautsch...another simply amplify the vector and insert with separate primer pairs in the same PCR. Add homologous sequences...

...therapy had fallen - plummeted really -  from grace 2 years prior with the death of Jesse Gelsinger in an... the whole field to a grinding stop and only now, 2 decades later, is it finally back in the limelight...the other hand, have broad tropism in the eye. AAV-2, the first identified serotype and the one used in...reading 1. FDA approves gene therapy for blindness 2. Gene Therapy emerges from disgrace to be the next...occurring animal model, the RPE65−/− dog, was characterized and found to suffer from early and severe visual...

... However, if you have a larger knock-in (200 bp – 2 kb), a dsDNA donor will need to be used due to synthesis...accidentally change your protein of interest.   Fig. 2: Stop re-cutting with PAM and guide RNA disrupting... the other repair mechanisms Mammalian cells have 2 primary repair mechanisms besides HR - Non-homologous.... Com., 8(2024), (2017). 10.1038/s41467-017-01836-2 Charpentier, M., Khedher, A. H. Y., Menerot, S., Brion...recognition sequence in the donor DNA, you can guarantee there won’t be re-targeting after HDR. If your...

...indicated they wanted to see more negative data (Figure 2.) At the same time, when scientists who support open...is not only housed, but encouraged.    Fig. 2: Results from a survey conducted by Addgene in 2021...detailed methods and image descriptions, a minimum n = 2, and well labeled high- and low-magnification images...gene therapy trials. Human Gene Therapy Methods, 26(2), 71–76. https://doi.org/10.1089/hgtb.2015.040 Initiative... in Adeno-Associated Virus Serotype 9 (AAV9) Preparations. Human Gene Therapy Methods, 30(6), 214–225....

...create the 2nd-generation lentiviral system (Figure 2). The HIV genes that do remain are very important ...1996 Apr 12;272(5259):263-7. PubMed PMID: 8602510. 2. A third-generation lentivirus vector with a conditional...CH, Humeau LM, Rebello T. J Gene Med. 2013 Feb;15(2):78-82. PubMed PMID: 23322669. 6. Genetic treatment... Banasik MB, McCray PB Jr. Gene Ther. 2010 Feb;17(2):150-7. PubMed PMID: 19847206. Additional Resources...Mulder GJ, Polack A, Bank A, Soulier J, Larghero J, Kabbara N, Dalle B, Gourmel B, Socie G, Chrétien S, Cartier...

... to DFHBI in the presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more...PMID: 25713381. PubMed Central PMCID: PMC4364232. 2. Baker, Monya. “RNA imaging in situ”. Nature Methods...emerging environmental pollutants.” Front Chem. 2014; 2: 41. PubMed PMID: 25019067. PubMed Central PMCID: ...biosensors.” TrAC Trends in Analytical Chemistry 27(2):108-117 · February 2008. 11. Svensen, Nina and Jaffrey...would bring the relevant ends together rather than separate them (Fig. 1b). One can also make “signal-off”...

...droplet in each well (Figure 2). A clean ddPCR should have a clear separation between positive (blue) and... to 2 minutes and the denaturation time to 1 minute has been shown to increase droplet separation (Witte... qPCR is that the results can vary by a factor of 2. This means that if you set up two identical assays...getting a clean separation between positive and negative droplets with these parameters, here are a few...Since Addgene began producing AAV vector preparations, we have used quantitative PCR to evaluate the ... for ddPCR set-up. Prepare the master mix in a separate area from where you prepare your sample dilutions...your PCR For a good starting place on what PCR parameters to use, see Lock’s seminal paper on AAV titration...

...3 plates at different densities for selection). A 2% agarose solution is then melted (and thereby sterilized...96-well assay plate. We generally freeze at least 2 ampules each of 4 clones with different levels of ...of agarose, which is then overlaid with the firmer 2% agarose gel solution discussed above for picking....concentrations of salts and glucose. If we circled well-separated clones containing more than 50 cells (visible ... should contain at least 50 cells and be well-separated. If the growth rate/transfection efficiency is... with this!) Make sure your colonies are well separated. For this, you need to have some idea of the rate...individual cell clones with different expression characteristics from a population of transfected clones. The...

...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...phosphorylated intermediate of a protein for characterization. However, phosphoproteins are often low abundance...strategies; expression should be optimized for parameters such as induction temperature and length of induction...

...mammal called alpaca could neutralize the SARS-CoV-2 virus. But while the early results were promising,...receiving dozens of requests every day.    Figure 2: When a researcher needs a collaborator for a specific...Crowdfight will: 1) Find an expert with the right skill; 2) Confirm the expert’s suitability and availability...diseases, so we were left with no choice but to quarantine in our houses, feeling helpless about the situation...

...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...remain bound to each other until you are ready to separate them. Most proteins are not likely to remain attached...

... Jan;20(1):81-89. PubMed. 2. Bauer DE, Kamran SC, Lessard S, Xu J, Fujiwara Y, Lin C, et al. An erythroid...with TALENs, in collaboration with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore... inferred based on correlative biochemical characteristics and ectopic reporter assays. We have been interested..., identifying these deletions, and finally characterization. Read and download the full protocol from ...Dass A, Yien YY, Chung J, Masuda T, et al. Characterization of genomic deletion efficiency mediated by...

...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...multiple separate wells (within the same plate) with purified plasmid from the same aliquot/preparation. The...co-transfected with the test plasmid or transfected into a separate well of your cells. Regardless, the amount of ...should be consistent over time and from different preparations of the purified test plasmid expressing the ...Using our example above, we would test different preparations of Plasmid A in different aliquots of cells ...

... is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...per plate) across 20 fresh selective plates. Step 2: Express random phage DNA fragments in E. coli Now...microbiology 23.4 (2015): 185-191. PubMed PMID: 25708933. 2. Cooper, Callum J., Mohammadali Khan Mirzaei, and ... biology 34 (2015): 69-77. PubMed PMID: 26275959. 2. Weigele, Peter, and Elisabeth A. Raleigh. "Biosynthesis... to be explored and exploited. With better characterization, these proteins could function as surrogate...surrogate antibodies for identifying and characterizing sugars and sugar-containing molecules in molecular biology...colonies of interest, sequence the inserted DNA and characterize your new putative host cell-binding proteins...

.../10.1038/s41587-024-02268-2 Schep, R., Brinkman, E. K., Leemans, C., Vergara, X., van der Weide, R. H.... flanking sequences to their input string (Figure 2). This string can then be used as input for the prediction...top 5) and test them in their labs. Figure 2: Guidelines to predict pegRNA efficiencies with pridict.it...we can evaluate tens of thousands of designs in parallel. To do this, we performed high-throughput ‘self-targeting...the TRIP (Thousands of Reporters Integrated in Parallel; Akhtar et al., 2013; Schep et al., 2021) method...Assayed by Thousands of Reporters Integrated in Parallel. Cell, 154(4), 914–927. https://doi.org/10.1016...., Benvenuto, E., Schmidheini, L., Schep, R., Damodharan, T., Balázs, Z., Janjuha, S., Ioannidi, E. I....

...upstream of an opsin gene (e.g., channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry) to...the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable strong... Abdallah, Khaled, et al. "Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents...Deniz, et al. "A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range ...vectors to answer this question, mapping and characterizing the activity of the DA neurons of the VTA. ...brain area, demonstrating that it is possible to separate neuronal inputs to induce aversion- or reward-...PMC4971574. Hooper, Andrew, and Jamie Maguire. "Characterization of a novel subtype of hippocampal interneurons...

...) 38 kb Cells HEK-293T Sf9 Time to collection 2–4 days 5–10 days Purification Ultracentrifugation...Centrifugation Titration time ~1 day (AAV); ~1 week (lenti) 2–10 days Initial titers Higher Lower Resources... a factory to produce adeno-associated virus type 2 vectors. Human Gene Therapy, 13(16), 1935–43. https...transfection). This differs from typical insect cell preparations, which we talk about below. Processing The viral...Plasmids and Resources References Shaw, G., Morse, S., Ararat, M., & Graham, F. L. (2002). Preferential transformation...