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...Digest your DNA Set up restriction digests for your donor and recipient plasmids. Because you lose some DNA...plenty of starting material. We recommend 1.5-2μg of donor plasmid and 1μg of recipient plasmid. It is also...

...requirements are different for the donor and acceptor fluorophores. The donor must be delivered to an exact... when donor and acceptor are close together. The binding of acceptors >100 Å away from the donor doesn...Let’s consider the donor first where labeling specificity is most important. Donor labeling and the need...for specificity Donor fluorophores need to go to the right place. Mis-targeted donors never “see” FRET...by a sea of uncoupled donors. The best method for targeting a fluorescent donor to your protein is to ...Madani et al., 2009). Donor and acceptors: Putting it all together The FRET donors and acceptors described...table below for donor and acceptor pairs that you can use in your FRET experiments. Donor Acceptor Tag...

...done through the design and use of a donor DNA molecule. Your donor molecule will need to have homology...distance. Homology arms and types of donors To make sure your donor molecule is used as the template, you... a dsDNA donor will need to be used due to synthesis limits of oligonucleotides. These donors traditionally...homology-directed repair pathway, designing the best donor DNA, and avoiding common mishaps in this class of...sequence. This homology will guide the cell to use the donor DNA as a template for repair, which will result ...your sequence, a.k.a a knock-in! So how does the donor get used as a template for repair? Read on to find...break by homologous recombination.    Designing donors for HDR The basis of the HDR pathway relies on ...

...excited donor fluorophore to the acceptor fluorophore. The typical steps in FRET are: 1. Donor fluorophore...of a photon 2. Energy transfer from the excited donor directly to the acceptor fluorophore--think of it...FRET to occur several constraints must be met. The donor and acceptor fluorophores must be compatible, such...such that the emission spectrum of the donor fluorophore overlaps with the excitation spectrum of the ...fluorophore. Otherwise the energy transferred from the donor will not be able to excite the acceptor. In addition... efficiency of FRET can be measured for a given donor-acceptor pair and a change in FRET efficiency correlates...constructing intermolecular FRET probes, where the donor and acceptor fluorophores are fused to two separate...

...large donor templates using two sequential homology directed repairs (HDR) with CRISPR/Cas9. Donor A contains...after the gRNA target site. Donor B contains: 400 bp homology arms to donor A,and “part B” of the transgene...vector strategy for increasing the size of the AAV donor template for CRISPR-mediated homology directed repairs...Trans-splicing Overview: For trans-plicing, splice site donor and acceptor sequences are used to reconstitute ...fragment of the transgene, followed by a splice site donor. The AAV encoding part B of the transgene contains...muscular dystrophy. Three different splice site donor and acceptor sequences were tested and with the ...promoter, the 5’ half of the transgene, a splice donor sequence and the region of homology. The AAV encoding...

... the donor is quenched when FRET occurs. By photobleaching the acceptor, you release the donor's quenching...fluorescence decay time of the donor. When FRET occurs between the pairs, donor fluorescence is quenched and...non-radiatively from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) by means ...orientation will be fixed. The quantum yield of the donor (number of emitted photons per absorbed photons)...signal you should choose the highest quantum yield donor, the highest absorbing acceptor and fluorophores... the acceptor with the laser used to excite the donor. In that case the cross-talk is high and your background...one is the sensitised emission method, where the donor is excited by a specific wavelength of light and...

...using a donor-plasmid, you could use a single-stranded oligodeoxynucleotide (ssODN) as the donor [6]. The...transposase, which facilitates direct integration of the donor-transposon (carrying your cargo of interest) into...achieved by utilizing a piggyBac™ transposon in a donor plasmid carrying the DNA edit of interest, along...double-stranded DNA break. After DNA cleavage, the piggyBac™ donor is used by the cell’s host machinery for DNA repair...excision, but defective for integration [4], the donor cassette is removed leaving behind no accessory ...possible random re-integration from the piggyBac™ donor, which is typically found with the wild-type piggyBac...piggyBac™ transposase enzyme [4]. By using a donor plasmid containing a negative selection marker such ...

...gRNA or PAM site is disrupted in the donor. Choosing a type of donor: plasmid, oligo, double or single ...then displaces one strand of the homologous DNA donor and can template repair off the other strand; this...methods are characterized by use of a homologous donor (e.g., sister chromatid, plasmid, etc.), and consists...prone mechanism that notably does not require a donor template and thus won’t be covered in detail here...enzyme, a guide RNA to your site of interest, and a donor DNA molecule containing your desired edit are all...designing a repair template to create mutations HDR donors can contain insertable sequences or specific mutations...the biggest determinant when selecting a type of donor. ssDNA templates (referred to as ssODNs (oligodeoxynucleotides...

...double stranded donor DNA with other design parameters. Example single stranded donor DNA and a presumptive...replacement in vivo. We discovered that single stranded donor DNA: Complementary to the released strand, 127bp...presented in Figure 2. Figure 2: Single-stranded donor DNA interaction with the Cas9-DNA duplex. Image ...extremely high frequencies of genome cutting and, when donor DNA is included in the nucleofection reaction, extremely...expressed Cas9 protein, transcribed sgRNA, and optional donor DNA. Chris Richardson is a Postdoctoral Researcher...active and inactive CRISPR-Cas9 using asymmetric donor DNA." Nature biotechnology (2016). PubMed PMID: ...

.... Designing the donor plasmid Figure 2: Donor plasmid design strategy. A donor plasmid contains... key feature of our methodology is the use of a donor plasmid that contains long stretches of DNA on either...cut, we trick the cell into using the introduced donor plasmid as a repair template. This results in the...protein and the crRNA-tracrRNA complex) and the donor plasmid into the cells we use electroporation, a...design described above can be used to create similar donor plasmids for introducing tags into your gene of ...

...obtained as a universal donor plasmid. The CRISPaint toolkit includes several donor plasmids for protein ...arm repair template. SapTrap includes a prebuilt donor plasmid library containing several types of fluorescent...between the tag and homology arms). Digestion of the donor plasmids with SapI allows the tag, selectable marker...connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type will produce the same ...overhang, different combinations of connector and tag donor plasmids from the library can be used to generate...Auxin-Inducible Degron Tagging with Short Homology Donors.” Cell Rep.15(1) (2016):210-8. PubMed PMID: 27052166...

...using ssDNA oligo as donor template, it's usually more efficient than plasmid donor. Typical design is ...notes below are considerations for designing HR donor. Generally if off-target (non-specific) cleavage...then select the best guide to co-transfect in HR donor. Alternatively, a new system described recently ...insertions or deletions), we typically use plasmid donor, with two homology arms on each side flanking your... we use around 400 ng of a typical size (~5 kb) donor plasmid with Cas9/sgRNAs vector, for one well in...make mutations of the HR template to avoid this donor plasmid being degraded by Cas9 in cells. One good...or avoid putting in the full target site in the donor. For example, choosing target sites that span the...

... power of ssDNA donor molecules for homology directed repair. Using long ssDNA donors, the Gurumurthy ...ameliorate this problem. Briefly, you design your donor template to include a T7 promoter upstream of the...’ll need to design only a single guide and your donor ssDNA. Here, we’ll use the example of inserting ...previously made mouse model. To avoid disrupting splice donor/acceptor sites, Miura et al. (2018) recommend targeting...49%). In contrast, Easi-CRISPR with a long ssDNA donor was less efficient using i-GONAD (15%) vs. microinjection...conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.” Genome Biol. 18...conditional knockout mouse models using long ssDNA donors.” Nat Protoc. 13 (2018) (1):195-215. DOI: 10.1038...

...sgRNA that targets the donor vector sequence. Homology arms that flank the donor plasmid cargo are complementary...from Jeffrey Essner’s lab uses CRISPR/Cas9 and a donor vector to create knock-ins of fluorescent proteins...the cargo (ex: fluorescent protein gene) on the donor vector. Therefore, targeting requires two sgRNAS...

...sites flanking the insert and the attP sites of the donor vector. This reaction is catalyzed by the BP Clonase...the reaction, the ccdB gene is excised from the donor vector.   Figure 2: The Gateway system adopts... TOP10, Mach1). The ccdB gene is present in the donor vectors and the destination vectors prior to recombination... of an attB-PCR product or plasmid with an attP donor vector. In this case, we would use PCR to add attB...attB-flanked PCR product with an attP-containing donor vector.   Method B: TOPO-cloning of the desired...vectors Vector type  Vector features Purpose Donor vector attP sites for recombination; ccdB gene for...

... , EGFP-ShadowY λ Dex : Donor maximum excitation wavelength (nm), QY D : Donor quantum yield, λ Aem : ... is non-radiatively transferred from an excited donor fluorophore to an acceptor. Because this process...fluorescence microscopy or spectroscopy by exciting the donor and detecting the emission of the acceptor. Alternatively...the fluorophore and emission of a photon) of the donor can be measured using fluorescence lifetime imaging...increased energy transfer through FRET reduces the donor lifetime. FRET is often used to study (1) protein-protein...interactions where each protein is separately fused to a donor or acceptor molecule (also referred to as intermolecular...conformational changes within a protein where the donor and acceptor are both fused to the same protein ...

... targeting the genomic site and the donor vector. The PITCh donor plasmid with an EGFP-2A-PuroR cassette... and donor vectors as well as pX330S-2-PITCh, expressing a generic gRNA targeting the PITCh donor plasmid...Plasmids Protein Species Tag Donor Plasmid gRNA plasmid gRNA plasmid FLAG pFETCh_Donor CEBPB Human FLAG pFETCh_CEBPB...may be used. The HDR (homology directed repair) donor plasmid with homology arms and selection marker....as design and clone in the homology arms for the donor plasmid. The Mendenhall and Myers labs have also...instead of HDR, which makes it easy to construct a donor vector, because MMEJ utilizes very short (around..., & FBL-specific gRNA pCRIS-PITChv2-FBL - PITCh donor vector for EGFP-2A-PuroR knock-in into human FBL...

...fluorescent donor for a bioluminescent donor, making BRET probes. Here the bioluminescent donor is unaffected...exciting the most blue-shifted fluorescent protein (donor) and measuring the resultant emission from the red-shifted...autofluorescence, and, in the case of exciting cyan-excitable donors, phototoxicity. Another challenge to using FRET...wavelengths of light to initiate signaling. When FRET donor excitation wavelengths overlap with optogenetic ...fluorescent protein acceptor. NLuc is an excellent BRET donor due to its bright luminescence. Intracellular Calcium...

...Adenoviral CRISPR vectors (left) or CRISPR vectors and donor DNA vectors (right) are transduced to target cells...non-homologous end joining (NHEJ). In targeted DNA knock-in, donor DNA is copied into the cut site through error-free...RNA-guided nucleases or TALENs together with AdV donor DNA leads to a vast majority of AdV-modified human...structures presumably reduce the likelihood that donor DNA will interact with sporadic double-stranded ... via simple switching of the relevant gRNAs and donor DNA Adenoviral CRISPR/Cas9 genome editing tools ...

...transferred from a donor bacterium to a recipient bacterium through direct contact. The donor bacterium contains...integrated within a bacterial chromosome and allows the donor bacterium to make a small “bridge” or sex pilus ...recipient cell drawing it close. Once in contact the donor can transfer genetic material to the recipient bacterium...