... used with a wide variety of restriction enzyme-based cloning methods. Each method has its own pluses ...outside of their recognition sequence, creating four base flanking overhangs. Since these overhangs are not... original plasmid (Option 1) or added using PCR-based amplification (Option 2). Advantages of Golden ...Golden Gate cloning is its scalability. Unique 4 base overhangs can be used to assemble multiple fragments...advantages over other cloning methods. Exonuclease-based methods like Gibson assembly require 20-40 bp of...DNA fragments at a time. As with all Golden Gate-based methods, this system exploits the ability of Type...Bogdanove and Voytas groups described a new Golden Gate-based technology for genome editing which allowed for ...

...DU al L uminescence-based C o- I mmunoprecipitation) plasmids : Luminescence-based protein-protein screening...Lu minescence‐based T wo‐ Hy brid) : Combines in vivo BRET with ex vivo luminescence-based co-immunoprecipitation...Scientists have developed a wide range of luciferase-based plasmid systems for many different applications....NanoLuc® complementation : Protein-protein assays based on a split NanoLuc® have been used to assess GPCR-G...Antares and Enhanced Nano-lanterns) to allow for BRET-based in vivo imaging of multiple cellular processes, ...

...CRISPR plasmids based on Cas13 designed to precisely edit RNA targets....Browse CRISPR Plasmids By Function Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation ...

...therapeutic potential for the split base editors. Find the split base editors at Addgene. Levy et al., Nature...please sign up here.        AAV delivery of split base editors Article contributed by Jennifer Tsang AAVs...limits the delivery of larger transgenes, such as base editors which have a gene size of ~5.2 kb. To overcome...David Liu’s lab developed split cytosine and adenine base editors that can be reconstituted in vivo to form...sequence to the unmodified enzyme. Each half of the base editor is fused to a fast-splicing split intein ...peptide bond at the split site. Figure 1: The base editor is split into two plasmids and co-transfected... a single AAV expressing GFP. The lab tested the base editors in vivo and achieved therapeutically relevant...

...Biolabs offers non-toxic protein labeling systems based on SNAP-, CLIP-, ACP-, and MCP-tags for visualization...-tag, and GFP . Technology SNAP-tag SNAP-tag is based on the human O6-alkylguanine-DNA-alkyltransferase...MCP tags ACP- and MCP-tags are small protein tags based on the acyl carrier protein. Substrates are derivatives...

...applications that lend themselves to aptamer-based instead of protein-based fluorophores, including: Environments...course of the next two decades, these nucleotide-based ligand binders would prove to be highly adaptable... few important advantages over their amino acid-based counterparts, antibodies and enzymes, such as: ...many entirely new applications including aptamer based fluorescent biosensors. Aptabeacons Fluorescence...instance, the fluorophore and quencher may form the base of the stem in an RNA stem-loop structure (see Fig...compound malachite green (MG) to create a non-protein based visualization technique. Ultimately, MG proved to...eukaryotes, the Jaffrey lab has since developed new RNA-based scaffolds to contain and stabilize Broccoli cassettes...

...The first base of the 3′ extension of the pegRNA should not be C. A C base is speculated to base pair with...begins with a C base can cause unwanted base pairing (left), while pegRNAs that use other bases in this location...near point mutations to create 3-base (or longer) tracts of edited bases. DNA mismatch repair (MMR) is less...efficiently targets single-base mismatches (left), but “bubbles” of 3 or more mismatched bases can often evade MMR...efficient at identifying these “bubbles” of mismatched bases, enhancing correct editing efficiencies (Chen et... create linkers that minimize unwanted intra-RNA base pairing with the primer binding site (Nelson et ...

... than base editing (in certain circumstances) Current base editors can perform any base-to-base conversion...12 possible base-to-base changes in a single edit. Prime editing is also more precise. Base editors, for...targeted insertions, deletions, and all possible base-to-base conversions (Anzalone et al., 2019). Plus, it...can be over 30 base pairs for prime editing. Since PAM sites occur on average every ~8 base pairs on either...DNA strand, many previously developed base editors with a <8 base-pair editing window cannot edit within... example, typically edit all the target bases within the base editing window, while prime editors make...nucleotides are positioned within the canonical base editing window, base editing has higher efficiency and fewer...

...widely adapted for a wide variety of applications: base editing, CRISPR activation and inhibition, among...visualization tools is CRISPR-Sirius, the brightest CRISPR-based tool to date for visualizing genomic loci in living... Massachusetts Medical School developed a dCas9-based multicolor labeling system to visualize chromosomal...St1Cas9. These fusions localized to specific loci based on the guide RNA sequence. A year later, they developed...et al., 2018). They improved on previous CRISPR-based loci visualization tools by increasing gRNA stability... aptamer insertion in the gRNA tetraloop (a four base hairpin loop motif within the gRNA) resulted in ...loci gave a detectable signal in human U2OS cells. Based upon this, they chose an intergenic DNA region (...

...which kit to use for your experiment. Bead-based or column-based kits — which to choose? Plasmid purification... column-based DNA purification methods perform well in terms of quality and yield. Bead-based purifications...illustrates the three grades of DNA you may need based on their relative quality and quantity.   ...1: Plasmid preps fall into different categories based on their relative qualities and quantities. Image...eluting the DNA from the substrate. In a column-based approach, the substrate is a silica matrix fixed... usually by centrifugation or vacuum. In a bead-based approach, you mix specially coated magnetic beads...ultimate plasmid yield.  Take your average column-based miniprep kit for example. These kits are designed...

...the black plague).  Base editors The major drawback of all the endonuclease-based gene editors (meganucleases...enzyme to generate the prototypical base editor enzymes. The cytidine base editors (CBE) use a cytidine deaminase...understanding of the adenine base editors which can help accelerate the engineering of novel base editor proteins....movie’s name is itself a combination of the four bases of DNA – A, T, G, and C. In this movie Ethan Hawke...several ZFNs are strung together, it can read many base pairs simultaneously to increase specificity. This...as CRISPR-Cas editing soon replaced the protein-based DNA-binding domain with a single-guide RNA sequence...the genome. This was soon followed by the adenine base editor (ABE) enzyme in 2017. ABEs rely on a similar...

...CRISPR-based approaches, several groups have recently worked to enhance the efficiency of AAV-based gene...gene is removed via infection with adeno-cre. AAV-based gene editing compared to CRISPR editing CRISPR, ...more challenging to develop high efficiency CRISPR-based approaches for the generation of knockins via homologous...However, there are important disadvantages to the AAV-based approach that must be considered when deciding whether...experimental approach. First, unlike CRISPR, the AAV-based approach is only able to target a single allele ...the same cell, are more appropriate for a CRISPR-based approach. Second, the AAV preps generally used for...in a given experiment. For this reason, the AAV-based approach requires G418 selection of infected clones...

...when working with lentivirus: these viruses are based on HIV-1, which may require additional lab biosafety...A Envelope VSV-G recommended for the FELIX (FIV-based) system Nolan 22502 pCEP4-tat N/A Packaging Expression... Puro selection. Shih 25737 pSLIK-Hygro 3rd Tet-based inducible shRNA or cDNA expression, gateway plasmid...mir-RUP-PheS 3rd Lentiviral shRNA plasmid (pGIPz based) for inducible knockdown, multicistronic design ...transfer plasmid are size (i.e., a limited number of basepairs of nucleic acid can fit into a viral particle)...

...generation sequencing. Unlike absorbance-based methods, fluorescence-based methods require a standard curve, ...contamination based on the presence of other bands or streaking. Unlike absorbance based methods, this...ways to do this and the method you choose could be based on your downstream application, time, and instrument...correct size. This is something that absorbance based methods can’t tell you and you don’t need a spectrophotometer...of your DNA at different dilutions and quantify based on band intensities of your DNA relative to the ...plasmid prep. Diphenylamine method Another absorbance based method for quantifying DNA uses diphenylamine. Diphenylamine...

...  Listen to the base editing podcast segment The increasing popularity of single base-editors has resulted...plasmid that allows for rapid fluorescence-based quantification of base editors that utilize dCas9 fused to ...imaging capabilities. Upon developing a new imaging-based bacterial colony screening platform, Oliver Griesbeck...fluorescent reporter for rapid quantification of base-editing and Cas9 cleavage activity Article contributed...editing toolkit. However, a challenge in optimizing base editing technologies across several cell types is... human APOBEC3A-Cas9n-UGI and APOBEC3B-Cas9n-UGI base editing complexes are more efficient than the BE3...

... You can generally add any 6 bases, but you should ensure that the bases do not result in the formation...In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time...a recipient plasmid. Designing primers for PCR based cloning     The basic PCR primers for molecular...molecular cloning consist of: Leader Sequence: Extra base pairs on the 5' end of the primer assist with restriction...information). Thus, we recommend that you add 3-6 bases upstream of your restriction site to improve cutting...amplification. We can start similarly, taking the final 18 bases of the ORF, including the stop codon (5'-TGGCATATCTCGAAGTACTGA... is. You should select an annealing temperature based on the melting temperature (Tm) of the portion of...

...of hands-on time. Creating a modular, selection-based gene tagging approach Multiple features of C. elegans... the selection marker. Other labs have used PCR-based screening to detect mutants, but this method limits... sought to create a new and efficient selection-based CRISPR/Cas9 method to generate knock-ins on any ...Genetics Society of America. To use this SEC-based system, first design and clone 500-700 bp homology... candidate animals can then be further screened based on their Roller phenotype. If SEC is inserted at...animals that have undergone SEC excision are screened based on the loss of the Roller phenotype. These animals...attempt for 6/7 genes. The data are clear - the SEC-based system is both simple to use and highly efficient...

...traditional cloning methods. In traditional cloning, base-pairing in the short overlapping regions (usually...overhangs of varying length (typically 10-12 bp) based on a specific sequence. The annealed but nicked ...in the reaction, meaning that T4 Pol will remove bases from the 3' end of the cut site until the first ...only the 5' primer is shown here. Note: Use web-based primer design software to ensure a melting temperature...presence of dCTP (not dGTP), corresponding to the first base removed by the restriction digest to create the ...

...’, where N can be any base. In Figure 5, we can see why: two arginines make base-specific hydrogen bonds...stability for the next DNA base pair to be unwound. A mismatch within the 10-base seed sequence will destabilize...guide RNA and wondered why it is limited to only 20 bases, or why it’s so important to choose a target sequence...nucleotides 1-10 of the guide RNA, prearranged for base pairing with a complementary DNA strand. These 10...only bound by the phosphate backbone. These PAM bases must be present for Cas9 to unwind the DNA and check...Residues R1333 and R1335 from the CTD (yellow) make base-specific hydrogen bonds to guanines of the PAM in...complementarity with its guide RNA. But how to test DNA bases that are already snugly paired to each other? At...

...the content of our paper, “Synthetic recombinase-based state machines in living cells” (1), into a more...how to adapt our engineered devices, recombinase-based state machines for your own experimental needs. ...the order or timing of these factors. Recombinase-based state machines (RSMs), which take on a particular...particular DNA-sequence configuration (state) based on the identity and order of a particular set of inputs, ...Sanger sequencing, qPCR, and fluorescent reporter-based) each come with their advantages and disadvantages... the predominant state of registers in a sample based on a similarity metric that we defined in our paper...convenient strategy is the fluorescent reporter based method since it lends itself to cheap, quick, and...