...CRISPR to detect SARS-CoV-2, including many with plasmids deposited at Addgene! 2. DNA Purification Without...detection methods, synthetic nanobodies, SARS-CoV-2 protein expression, and more. We also highlighted ...empty backbones, fluorescent protein tags, SARS-CoV-2 plasmids, etc. It is our hope that by providing cloning...RNA and extracting RNA without a kit. 3. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas by ...multiple plasmids in one cell, it might be that you can't stably maintain them in the cell. This blog post features...

...: 24901218. PubMed Central PMCID: PMC4047077. 2. Pagliara, Patrizia, et al. "Ostreopsis cf. ovata induces...times), depending on the cell line so old cells should be retired! Conversely, cells younger than the 3rd ...Make sure cells don't stay out too long While you’re preparing things, make sure your cells don’t stay...contaminants. Set up a routine to split cells Don’t be lazy about splitting cells, instead try to form a routine...’s best to split cells before the plates are more than 80-85% confluent because cells like their space...lactic acid (a by-product of cellular metabolism that is toxic to the cells), and need to be split very...numbers! When starting off with a new cell line, grow the cells, aliquot them into a number of vials, ...

...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the...but these cells are prone to mutations and will have more unintended changes to their genomes. 2) Designing...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required for recombineering...generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering system...pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically ~70 nucleotides...bacteria that has key MMR proteins knocked out or 2) specially design ssDNA oligos to avoid MMR: 1) E....

... York: Crown Publishers, 2010. 2. Gold, Michael. "A conspiracy of cells." State University of New York...quality work in cell culture. The problems with cell lines Simply put, the majority of cell line problems...HeLa cells, the first immortalized human cell line. He proved this by examining whether the cell lines...Cancer Cells, Marker-independent identification of glioma-initiating cells. The International Cell Line ...working cell bank has been made by a research institution (an excellent practice), or maybe cells have been...Animal Cell Culture Guide Thermofisher Guidelines for Maintaining Cultured Cells Invitrogen Cell Culture... Outreach Scientist Nick Amiss. Cell culture in the present day Cell culture is a widespread tool used...

...electrocompetent cells is minimal (picogram range). It can be extracted from small bacterial cultures (i.e. 2-3 ml...ratios measured at 260 and 280 nm should fall ~1.8 - 2 and give scientists an indication of purity versus...contaminants. The 260 and 230 nm ratios should fall ~2 - 2.2, and provide an indication of purity versus ...hydrochloride used during plasmid extraction. Figure 2: Use cloning grade DNA for direct cloning of your ...lipopolysaccharide of the cell wall of Gram negative bacteria like E. coli that affects tissue culture cell line viability...introduces transformation grade plasmids into a bacterial cell. Transformation grade DNA  Transformation is ...Figure 3: Need to introduce plasmids into mammalian cells? Use transfection grade DNA. Transfection is ...

...GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...challenges in 2-photon in-vivo imaging of voltage fluctuations. Image credit: Liu Z, et al. Cell. 2022.  ...suitable for in vivo optogenetic use.   Fig. 2: A) HcKCR1 is highly selective for K+ over other Na...slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies...two-photon microscopy. Cell, 18, 3408 - 3425.e29. (2022). https://doi.org/10.1016/j.cell.2022.07.013.   ...specific for human c-myc proto-oncogene product. Mol Cell Biol. Dec;5(12):3610-6.(1985). doi: 10.1128/mcb.5.12.3610...

...Table 2 for more details on the effect of pH on the fluorescent properties of Rosella.   Table 2: Effect...in yeast. Autophagy,4(2), 205-213. doi:10.4161/auto.5331. PubMed PMID: 18094608 2. Sargsyan, A., Cai, J...environment like the vacuole or lysosome. See Table 2 for a summary of what color fluorescence Rosella emits...fluorescence accumulates in the vacuole. See Figure 2 for an example of what fluorescence looks like for... Mammalian Autophagy Research. Cell,140(3), 313-326. doi:10.1016/j.cell.2010.01.028. PubMed PMID: 20144757...analyzing autophagy of cytosol and organelles in yeast cells. Autophagy (Greek for “self-eating”) is induced ...radio-labeled long-lived proteins as a proxy for autophagy. Cells are cultured with isotope-labeled amino acids for...

...Journal of Cell Biology. PubMed PMID: 29263082 Find plasmids from this publication at Addgene. 2. Bates,...antibody has an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are big enough...enough that multiple labels (green circles in figure 2 below) can be attached to each antibody. But being...displacement Require separate incubation of 1° and 2° Must use different species and/or different IgG ...Greater resolution due to lower label displacement 2° incubation can be skipped by pre-binding to 1° ...a fluorescent tag and the target antigen to about 2 nm, which can lead to higher resolution for super-...subclass.   Express the Nanobody In Bacteria: Table 2 also has a link to the plasmid you’ll need to express...

... a range of reasonable MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially if you’re doing...(Figure 2) and I was surprised at how variable Cas9-expression was across the different cells (Figure ...Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but I didn’t know what...knockouts and knockins in human somatic cells. Nature Protocols 2007; 2(11): 2734-46. PubMed PMID: 18007609...despite having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity using this assay, you really...Western blotting against GFP expression, but I didn’t. 2. In the original publication they see GFP expression...and I got clear bands using the conditions above. 2. Design your primers so that the digestion site isn...

...different steps and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail,...stimulation         In the video above, Channelrhodopsin 2 (Chr2) has been expressed in the motor cortex of a...lasers are relatively cheap and will run you about $2-3,000/laser. However, an important caveat is that ... the light-gated cation channel, Channelrhodopsin 2 (ChR2), exists in several states: closed/light receptive...PMID: 21628464; PubMed Central PMCID: PMC3138296. 2. Sparta DR, et al. Construction of implantable optical...board, Jacqueline N Crawley  [et al]. 2012;Chapter 2:Unit2 16. doi: 10.1002/0471142301.ns0216s58. PubMed...Pharmacology and Optogenetics. Cell 162.3 (2015): 662-674. doi: 10.1016/j.cell.2015.06.058. Pubmed PMID: 26189679...

...eGFP tag, and 2) gRNAs Number of loci targeted One Many Sample type(s) Cell lines Cell lines, primary...Expression system Stable cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or more...Originally published Dec 7, 2017 and updated Jul 2, 2020. Promoters may be the star of gene regulation...sequence targeted in the experiment. Figure 2: Comparing CAPTURE and CAPTURE 2.0.     Original...identified and the level of on-target enrichment. 2. Determining the spatial and hierarchical organization...Cytogenetics, 11. https://doi.org/10.1186/s13039-018-0368-2  Liu X, Zhang Y, Chen Y, Li M, Zhou F, Li K, Cao H... of cells (~5x107). Together, these two requirements prevent the use of CAPTURE with primary cells or ...

...that have been developed into retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses...effectively. Retroviral structure   Figure 2: Structure of a retrovirus. Image re-used from Dong...Dong & Kantor, 2021 under CC-BY license. Figure 2 gives a detailed view of the structure of a retroviral...transfer of RT, which would actually occur in step 2 during uncoating. After uncoating, reverse transcription...one of the two strands’ DNA counterparts from step 2 will be integrated by the viral integrase protein ... Air. Environmental Science & Technology Letters, 2(4), 84. https://doi.org/10.1021/ACS.ESTLETT.5B00050...synthesized inside of what is called the “target cell” (or host cell) by the viral protein reverse transcriptase...

...reviewed in 2, 5). The minigenome is capable of being replicated and transcribed in the cell when the appropriate...study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a minigenome?   The EBOV ...nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola...as it would for a full-length viral genome (Figure 2). While it is technically possible to encode all four...500 ng NP, 500 ng VP35, 100 ng VP30, 100 ng L, and 2 μg minigenome plasmid per well (in a 6 well plate ...PubMed PMID: 9847328. PubMed Central PMCID: PMC103829. 2. Conzelmann, K. K. "Reverse genetics of mononegavirales...choose cell lines that are highly transfectable.  The amounts of each plasmid transfected into cells are ...

...University of Manitoba due to their Biosafety Level-2 or 2+. Please Note: Despite the fact that AAVs themselves...Location (1): What tissue? Location (2): Where in the tissue/which cell type? Location (3): Traveling from...in very specific cell populations. Find Ready-to-Use Retrograde AAV Preps at Addgene 2) Comparison of genetically...long and stable expression (up to 9 months in mice [2]). Low Immune Response: Compared to other viral vectors...may be sufficient to overcome the transport issues. 2. Failure to plan (for storage) Is planning to fail...Therapy 15.10 (2007): 1740-1743. PubMed PMID: 17882145. 2. Zincarelli, Carmela, et al. "Analysis of AAV serotypes... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain." PloS one 8.9 (...

...fluorescent state make them useful for 2-color imaging with green PA-FPs. Table 2: Properties of Selected Reversible...PA-FPs come in 2 flavours- monomeric and tetrameric. Tetrameric PA-FPS are better for whole cell imaging and... of proteins and cells." Science 297.5588 (2002): 1873-1877. PubMed PMID:12228718. 2.Subach, Fedor V.,... conventional FP imaging. PA-FPs are divided into 2 main categories - those with irreversible and reversible... forms of the chromophore which contribute to the 2 peaks of wtGFP’s excitation spectrum- a major 397 ... / N99K / S148L / I165V / Q167P / L169V / I203R) (2) and PA-mRFP1(S146H / I161V / I197H) (3) were derived...with two distinct wavelengths of light (see Table 2). The best known reversible PA-FP is the naturally...

... (details in [2]) and can be used to quantify rates of mutation in a bulk or single cell-derived genomic...resulting from non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation... have been reported for mismatch nuclease assays (2, 6, 7). Ability to distinguish between mono-allelic...assays, in addition to testing their performance (2). Remember that although digital PCR is a great screening...PMID: 24509632. PubMed Central PMCID: PMC4063274. 2. Findlay, S. D., Vincent, K. M., Berman, J. R. & Postovit... successfully analyzed gDNA from a few thousand cells from single wells of a 96-well plate without prior... that they are easily able to distinguish single-cell derived samples with mono-allelic mutations versus...

...Diverse Class 2 CRISPR-Cas Systems. Molecular Cell 60:385–397 . https://doi.org/10.1016/j.molcel.2015.10.008...Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi.org/10.1016/j.cell.2015.09.038 Additional... can be used to diagnose the presence of SARS-CoV-2 RNA, but inadequate access to reagents and equipment...named this system SHERLOCKv2 for SHERLOCK version 2. Here are some of the improvements: SHERLOCKv2 uses... a protocol for using SHERLOCK to detect SARS-CoV-2 RNA. The test is started using RNA purified from patient...recently adapted the DETECTR platform to detect SARS-CoV-2 using a lateral flow strip format. They published ...CY (2020) CRISPR–Cas12-based detection of SARS-CoV-2. Nature Biotechnology. https://doi.org/10.1038/s41587...

... of 2 components: 1) an SB transposase, the enzyme required for catalysis of transposition; and 2) a transposon...stable transgenic cell lines. Biotechnol J. 2015 Feb 4. doi: 10.1002/biot.201400821. PubMed. 2. Mates L, et... gene (red) for expressing the transposase enzyme.2.) The transposase enzyme is expressed (red stars) ...to infect and replicate in host cells, but they are susceptible to cells’ defense mechanisms. The use of... transposon; the plasmid is then degraded by the cell.4.) The transposase creates a double strand break...improve its gene insertion efficiency in primary cells. In 2009, Zsuzsanna Izsvak’s lab screened many SB...tranposase variants for hyperactivity in mammalian cells. Their efforts resulted in a new and improved SB...

...TUs. Level 2: Assembly of multiple Level 1 TUs into multigene constructs. Figure 2: The three ...can be queried by testing any number in the Level 2 assembly. These pathways can require a lot of optimization... of multigene constructs. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765.  Hahn,... (2020). https://doi.org/10.1186/s12870-020-02388-2 Resources on Addgene.org Addgene’s Modular Cloning...complex cellular behaviors requires making complex manipulations to cells. For example: in a cell there ... yield a cellular phenotype, translational output, etc., which can support a larger cellular engineering...TU, or even an individual part, will behave in a cell. For unpredictable cases, MoClo provides two answers...

...illustrated in the schematic below.   Figure 2: gRNA target sequences (colored rectangles) are cloned... plasmids: This plasmid set allows you to express 2-4 gRNAs, with four being the ideal number. First you...the total number of gRNAs you wish to clone, from 2-7. For example, if you’re expressing 4 gRNAs, you’...gRNAs into spectinomycin-resistant plasmids pX330S-2 to pX330S-(last gRNA number). The 5’ most gRNA is ...Multiplexing Plasmids: These plasmids allow you to assemble 2-4 gRNAs through Golden Gate or Gibson Assembly. gRNAs...Multiplex Plasmids: These plasmids allow expression of 2-5 gRNAs in zebrafish. Custom destination vectors are...and a subsequent curing protocol that requires only 2-3 hours incubation. Kondo Lab multiplexed base editing...