...identification.   Fig. 2: Gel image of Plasmid 61564: pBAMD1-2 (~4.7 kb) digested with SpeI and... at ~3.6 kb and ~1.1 kb (lane 3). Uncut DNA (lane 2) has a band at <3 kb, which corresponds to the supercoiled...formation of plasmid multimers. Cell. 1976 Dec;9(4 PT 2):707-16. doi: 10.1016/0092-8674(76)90134-3. PMID: ...dimers in Escherichia coli K-12. Plasmid. 1989 Mar;21(2):138-41. doi: 10.1016/0147-619x(89)90057-7. PMID: ...Plasmids and Chromosomes. Microbiol Spectr. 2014 Oct;2(5). doi: 10.1128/microbiolspec.PLAS-0025-2014. PMID...Behavior of Plasmid DNA. Microbiol Spectr. 2015 Apr;3(2):10.1128/microbiolspec.PLAS-0036-2014. doi: 10.1128...plasmid monomer from a plasmid sample, or if no monomers exist in your sample, you could try linearizing ...

...low expression, and everything in between (Figure 2). These smears tend to occur when you are looking ...based on your experiment. You can see from Figure 2 that determining where to place your gate based on...the dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing a range of antibody...plot in Figure 4 compared to the dot plot in Figure 2. Quantification using MFI makes it easier to identify...105 signal intensity. If your samples and controls were processed the same way, the GFP- population should... control (left) or with the sample (right). Negative and positive samples are clearly distinguishable ...designed your first flow panel, you’ll load your samples into the cytometer and see one of two results for...

...University of Manitoba due to their Biosafety Level-2 or 2+. Please Note: Despite the fact that AAVs themselves...long and stable expression (up to 9 months in mice [2]). Low Immune Response: Compared to other viral vectors... location. Location (1): What tissue? Location (2): Where in the tissue/which cell type? Location (3...may be sufficient to overcome the transport issues. 2. Failure to plan (for storage) Is planning to fail...Find Ready-to-Use Retrograde AAV Preps at Addgene 2) Comparison of genetically engineered receptors (i.e...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... 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... above background. The majority of PA-FPs come in 2 flavours- monomeric and tetrameric. Tetrameric PA-...most PA-FPs, but, in 2015, the Looger Lab designed 2 variants of EosFP that can withstand these conditions...

...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...

.... To understand why that is, let's look at Figure 2. The blue laser excites at a wavelength of 488 nm ... of PE's excitation spectrum (dotted line, Figure 2). The yellow/green laser, however, excites at 561 ... stronger — or brighter — signal.   Figure 2: Excitation and emission spectra for PE. The excitation...this example, I will describe a basic panel (Table 2, Figure 5) for a hypothetical experiment in the lab...efficiency (eGFP expression).     The first item in Table 2 is a standard and should always be included—the live...from the other fluorophores to avoid bleeding and 2) BV421 is a very bright fluorophore, which makes it...case the CD45 expression turns out to be weak. Table 2: A simple flow panel to detect live, transduced, and...

...extraction solution, runs the protocol, then uses around 2-4ul in each 50ul PCR reaction. The gDNA extracted ... similar to using a single chimeric gRNA. At the same time, the off-target effects are reduced because...genomic PCR, but couldn't amplify the EMX1 gene using same primer you used in the Science paper ( Cong et al...

...improving editing accuracy by preventing re-editing and (2) optimizing “cut-to-mutation distance”. Furthermore...also takes into account that HDR is rare, usually 2-5% of the edited cells in our hands, these combined...screen a few hundred clones to find the right one Step 2 – Edit the cells a second time using a repair template...Additional CRISPR Resources on Addgene's CRISPR Guide Page 2. Optimizing "cut-to-mutation distance"  Even though... wisely (and let them cut close).   Figure 2: Relationship between cut site to mutation distance...PMID: 23287718. PubMed Central PMCID: PMC3795411. 2. Mali, Prashant, et al. "RNA-guided human genome engineering... events were corrupted by unwanted indels on the same allele, probably due to concomitant NHEJ repair....

...for the 2+ million DHS sites. See below for further details. DNA is a 3D Fractal Figure 2: DNA compaction...: 1. Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the ...NANOG enhancers 3. CRISPR out the enhancers from step 2. See which ESCs die or differentiate Step 3 reveals...expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt these enhancers...to CRISPR out the enhancers we identified in step 2. The result is a heterogenous population of stem cells...lentiviral vector, an attenuated retrovirus in the same family as HIV. By design, our lentivirii are genetically...very-sensitive-local -L 9 -N 1 -U GRNA_23MERS -S GRNA_HITS.sam Many thanks to our guest blogger, Aneesh Karve...

...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...one of its exons has simply been replaced with the same sequence flanked by LoxP sites while the resistance...

.... 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... The glycerol in the buffer will make sure your sample settles in the gel well and the dyes provide a ...you to interpret the bands that you get in your sample lanes. Choose your ladder based on the expected...approximate the DNA mass of comparably intense samples of similar size. Run the gel at 80-150V until you...

...influx into the cell when stimulated. Channelrhodopsin-2 (ChR2), the first widely adopted optogenetic tool,... (Phy) interaction 660 Cryptochromes Cryptochrome 2 and CIB1 Blue-light–mediated induction of protein ...interactions based on Arabidopsis thaliana cryptochrome 2 and CIB1 450 LARIAT Inhibits protein function by reversibly...customizable TALE DNA-binding domain with cryptochrome 2 protein and its interacting partner CIB1 to control...transcription effectors OPTOSTIM PHR domain2 of cryptochrome 2 (Cry2) from Arabidopsis thaliana is fused to truncated... with single-spike temporal resolution. PLoS One. 2(3):e299. PMID 17375185 Kim B, Lin MZ. 2013. Optobiology...comparative analysis of microbial opsins. Nat Methods. 9(2):159-72. PMID 22179551 Saunders A, Johnson CA, Sabatini...

... to the lysosome where they are finally degraded (2, 3). Once you've created your fusion protein, it's...examples include the ImageJ plugins JACoP and Coloc 2, BioImageXD, and customized CellProfiler pipelines...colocalization analysis example is given in Figure 2 (adapted from 4) - Dunn et al. 2011 provides an in-depth...Since colocalization relies upon the detection of 2 independent fluorescent signals and their potential...here! References 1. Allen Institute Plasmid Pages 2. Cooper GM. 2000. The Cell: A Molecular Approach. ...The same way the human body is made up of organs, cells comprise compartments and structures, called ...insights about the proximity of two proteins within the same subcellular structure or protein complex. By coexpressing...

...indicates a virus containing the genome of serotype 2 packaged in the capsid from serotype 5. Use of these...neurons that are not efficiently transduced by AAV2/2, and is distributed more widely in the brain, indicating... pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism...PJ, Mandel RJ, Muzyczka N. Mol. Ther. 2004 Aug;10(2):302-17. PubMed . From virus evolution to vector revolution...toolkit for human gene therapy. Wu Z, Asokan A, Samulski RJ. Mol. Ther. 2006 Sep;14(3):316-27. Epub 2006...independently of DNA synthesis. McCarty DM, Monahan PE, Samulski RJ. Gene Ther. 2001 Aug;8(16):1248-54. PubMed ...

...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... practice to compare degradation rates between samples treated with or without an autophagy inhibitor....

...transductions and use a range of reasonable MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially...doing a screen. I made a few monoclonal lines (Figure 2) and I was surprised at how variable Cas9-expression...across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal cell lines generated... expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but I didn... appear green despite having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity using this...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...

.... The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people choose...overnight at 4 °C and their secondary antibody for 1–2 hrs at room temperature, which allows them to reduce...Tris-glycine 6–400 kDa Tris-glycine 100 V, 1–2 hours Easy and cheap to handcast Short shelf-life...–40 kDa Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours Good separation, quality, and stability ...separation of proteins of different sizes.  Table 2: Recommended gel percentages for various protein sizes...are consistent and stable at room temperature for 1–2 years and are available in formulas specific to your...response over a broad dynamic range that typically spans 2–5 orders of magnitude, which includes a wide dynamic...

...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...be at least 85% of the initial laser output. The same considerations apply to the polishing and preparation...

...plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from Li et al., ...1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed the replicon of pCas into a non-temperature-sensitive... pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella...MUCICAT better, it is faster. Using MUCICAT only needs 2 newly constructed plasmids (pDonor and pQCascade) ...rounds of transformation in 30 days. Figure 2: (A) MUCICAT plasmids for multicopy integration. (...our metabolic engineering case study of N-acetylglucosamine biosynthesis, we constructed a strain library...rounds of transformation to complete the N-acetylglucosamine strain library construction in 8 days. To ...

...testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two different ...) your experimental conditions and measurements; (2) the cells you’re targeting; (3) how much expression... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLOS ONE, 8(9),... Comparison of four promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1...analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral... different phenotypes of the same cell type, are intermingled in the same brain nucleus: a serotype that...tropism between animal species or even strains of the same species. What works well in a mouse may not work...