...digestion of the destination vector with SapI yields 2 sites- the first site accepts the sgRNA target sequence...marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type will produce... a gRNA vector that targets the gene of interest, 2) a plasmid to specify the reading frame of the insertion...power of this system lies in the ability of the endonuclease Cas9 to create a DNA double stranded break (...break at a specific genomic site and, using the endogenous homology directed repair pathway, a new user-...CRISPR has allowed scientists to tag and light up endogenous genes of interest to better understand normal...targeting vectors. These vectors can be used to tag endogenous genes and simultaneously introduce a selection...

...this year. Designing the donor plasmid Figure 2: Donor plasmid design strategy. A donor plasmid contains...location of the protein or structure of interest. Endogenous gene tagging is an ideal solution because it ...visualization of specific, individual proteins under endogenous regulatory control. But even with the advent ... and tested plasmids that use CRISPR/Cas9 to endogenously tag a wide variety of genes with GFP. These ...interest. Researchers have developed a variety of endogenous gene tagging systems. Addgene’s CRISPR-based ... introducing tags into your gene of interest. Endogenous gene tagging with fluorescent proteins as described...

...and Amin Hajitou. "Clathrin-mediated endocytosis and subsequent endo-lysosomal trafficking of adeno-associated...bioengineering 94.2 (2006): 322-329. PubMed PMID: 16528756. 2. Oh, Steve KW, et al. "Substantial overproduction ...fact, due to impaired endocytosis. In parallel assays designed to test the endocytosis of a fluorescent dye...cell membrane and are taken into the cell via endocytosis. In our study, we aimed to prevent the uptake...packaging cells by treating the cells with the endocytosis inhibitor sucrose. Previous work by Stoneham ...

... non-physiological, and difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several...Biol, 2003. 163(2): p. 257-69. PubMed PMID: 14581454. PubMed Central PMCID: PMC2173526. 2. Zacharias, D....structures including organized smooth endoplasmic reticulum (OSER) whorls. 2)A dimerizing protein of interest... nature are prone to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [3]....form dimers in a cell. The Kd of EGFP is 0.11 mM [2]. Following the simple logic outlined above, you might... designed to detect resulting in false positives [2, 4]. Solutions to the polymer problem There are some...Citrine) can be monomerized with an A206K mutation [2]. Addgene has many of these constructs and they are...

...status of MEK1/2 was tracked at 5 minute intervals via Western blot. Phosphorylation of MEK1/2 peaks at just... phosphorylated MEK1/2 levels, suggesting PP1/PP2A is responsible for this MEK1/2 negative feedback. ...PubMed PMID: 28232577. Figures 2-4 were modified from this reference. 2. Regot, Sergi, Jacob J. Hughey...Zhou et al studied the negative feedback on endogenous MEK1/2 phosphorylation by creating a short pulse ...precisely control the timing of kinase activity. 2. Studying negative feedback loops of signaling pathways...activity. They did this by exposing psRaf1 to a short 2 minute pulse of 500 nm light (turn on psRaf1) followed... Raf1, and CDK5 all behave similarly to their endogenous counterparts. Additionally, dimeric Dronpa is...

...repository of tools for SAR-CoV-2 research. Two new antibodies, Anti-SARS-CoV-2 Nucleocapsid Protein [mBG17...Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct epitopes on the SARS-CoV-2 nucelocapsid ...Anti-SARS-CoV-2 Antibodies here! Terry, J.S., et al. (2021). Development of a SARS-CoV-2 nucleocapsid ...prime editor PE7 Antibodies to facilitate SARS-CoV-2 research New viral vector preps of Voltron2 voltage... incorporates La’s N-terminal domain, PE7 (Figure 2).  PE7 shows enhanced editing efficiencies in a number...editor. Find PE7 plasmids here!     Figure 2: PE7 improves prime editing. A) Schematic of PE7 prime...PMID: 38570691. Antibodies to facilitate SARS-CoV-2 research   By Ashley Waldron Ready-to-use recombinant...

...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the...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....will have more unintended changes to their genomes. 2) Designing ssDNA oligos that avoid MMR activation:...

...Figure 1: Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Figure from Zetsche..., et al. "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed...., 2015. How was Cpf1 found and tested? Class 2 CRISPR systems, including the type II Cas9-based system...employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR from Prevotella and Francisella... the 3’ end of the target sequence. Figure 2: Effects of (A) double and (B) single base pair gRNA-target... Find plasmids from this publication at Addgene. 2. Zetsche, Bernd, et al. "Multiplex Gene Editing by...contain a RuvC-like endonuclease domain, but they lack Cas9’s second HNH endonuclease domain. Cpf1 cleaves...

...the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed to... through an experiment designed to knock out exon 2 of a given gene.   Figure 1: A knockout targeting...For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required, but 6 to 14 kb...construct to your target cells. After recombination, exon 2 of the targeted gene will be removed from the chromosome...the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted gene is disrupted (knocked out...recombination event will remove NeoR only and leave exon 2 floxed, as seen in line 4 of Figure 4. Since the loxP...cell which will recombine the template with the endogenous gene. Here, we'll describe the techniques and...

...fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced by proteolytic...fragments are produced by mild reduction of F(ab')2 fragments, which reduces the hinge region disulfide...Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common...advantages, though with some caveats.  First off, F(ab’)2 fragments are bivalent, so may have higher avidity...or Fab fragments. Higher avidity means that F(ab’)2 fragments may have overall stronger interaction with...Clinical Prospects of Antibody Fragments. Antibodies 8(2):28. https://doi.org/10.3390/antib8020028    Wessel...multiplexed super-resolution microscopy. Cell Rep Methods 2(10):100301. https://doi.org/10.1016/j.crmeth.2022.100301...

... F (2015) Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . ... 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... and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Molecular Cell 60:385–397 . https...

...and cryptochrome 2 (CRY2) (Zhang & Cui, 2015). When stimulated with light, the endogenous proteins activate...for controlling neuronal activity, channelrhodopsin-2 (ChR2), a blue light-activated cation channel, from...used in tandem with the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light ...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 References...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 Nagel...Hegemann, P., & Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane ...intracellular signaling pathways. Trends in Biotechnology, 33(2), 92–100. https://doi.org/10.1016/j.tibtech.2014.11.007...

... be extracted from small bacterial cultures (i.e. 2-3 ml) with or without commercially available kits ...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 ...these DNA samples must also be entirely endotoxin free. Endotoxin is a lipopolysaccharide of the cell wall...

.... (2015). Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell,163(3), 759-771... needing to work with a transfectable cell line. 2) They require larger expression vectors which are ...are also larger than their Cpf1 counterparts. Table 2. Cas9 Multiplexing Options  Multiplexing Method... Cpf1 expression vector.  See the graph in figure 2 for a comparison of editing frequency resulting from...Biotechnology 35.1 (2016): 31-34. Pubmed PMID: 27918548. 2. Zetsche, B., Gootenberg, J., Abudayyeh, O., Slaymaker... Components crRNAtracrRNACas9 crRNACpf1 Endonuclease size spCas9: ~4 kbsaCas9: ~3 kb ~3.9kb ...and doesn’t require the expression of other endonucleases, like Csy4, or the inclusion of processing signals...

...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   ... bacterial endotoxin is ensured to be less than five endotoxin units per mL. The endotoxin levels are ...recombination rates in FLEX/DIO/fDIO constructs. Endotoxin Endotoxin contamination in vector preparations can ...large animal studies. Endotoxin contamination is minimized by using an endotoxin-free plasmid purification...are determined using a chromogenic endotoxin detection assay based on the amebocyte lysate method. Purity...

... of 2 components: 1) an SB transposase, the enzyme required for catalysis of transposition; and 2) a transposon... gene (red) for expressing the transposase enzyme.2.) The transposase enzyme is expressed (red stars) ... 2015 Feb 4. doi: 10.1002/biot.201400821. PubMed. 2. Mates L, et al. Molecular evolution of a novel hyperactive...organism’s genome (see figure) and to disrupt endogenous genes at the site of insertion. In both of these...and binds the inverted repeats (green); and an endonuclease reaction occurs which cuts the DNA.3.) The released...

.... Diagnostic restriction digests are comprised of 2 separate steps: 1) incubating your DNA with restriction...which cleave the DNA molecules at specific sites and 2) running the reaction on an agarose gel to determine...including a 1.2 kb insert. The plasmid was digested with 2 unique enzymes (HindIII and BamHI) and run on an agarose...to serve as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed... JM110 or INV110. Avoid star activity. Some endonucleases (for example BamHI) are capable of cleaving ...manufacturers make High Fidelity versions of the endonucleases and/or supply custom buffers as means to avoid...

...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...

...repeated multiple times (2-4 rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification...creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the transgene...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...confirm its integrity. Initial production (2-3 weeks) - Here you’ll produce the primary recombinant adenoviral...media will turn yellow. Do NOT change the media (add 2-3 mL of fresh media once a week), and do NOT harvest...it will result in very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more HEK293 ...in a 10-100-fold increase in virus. Purification (2 days) - Purification is required if rAdV is to be ...

...difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away is suitable for ...factor 1 controls expression of transcription factor 2, which directly controls expression of a reporter ...modulating the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information. The ...our Hot Plasmids Podcast! TRIM-Away: Targeted endogenous protein degradation Article contributed by Mary... hr of treatment. In some primary cell lines, endogenous TRIM21 is sufficient for TRIM-Away, and you only... et al. Cell. 2017. PubMed PMID: 29153837. Endosomal pH sensors for STED nanoscopy Article contributed...contributed by Eric J. Perkins Listen to Endosomal pH Sensors podcast segment The first Addgene plasmid deposit...