We narrowed to 432 results for: sequence

...mutagenesis can be used to mutate the PAM sequence (an NGG sequence critical for Cas9 cleavage), thereby rendering...modification. Finally, the positive clones are sequenced to confirm the desired modification and the absense...Design As a rule of thumb, 11 bp of complementary sequence on either side of the desired mutation (usually... should be free of palindromic and repetitive sequences, but if present, a minor extension can usually...PCR primers can cause duplication of the primer sequence in the resulting plasmid. An additional restriction...duplications), final validation is performed by sequencing functional regions of the plasmid including the... kb (or are GC-rich and therefore difficult to sequence), it may be easier to subsequently clone a smaller...

...Should you cut it at the beginning of the coding sequence to delete the entire gene? Perhaps you are more...simple and easy. Several companies supply gRNA sequences, such as GenScript, ThermoFisher Scientific and...expression cassette reconstitution. When the target sequence was digested by sgRNA guided Cas9 endonuclease...bp genomic fragment containing the sgRNA target sequence can be placed in multi-cloning site (MCS) between...co-transfected with pX330 plasmids containing sgRNA sequences into HEK293T cells. The fluorescence intensity...develop primers to the approximately 500 base pair sequence surrounding your gRNA. Once amplified, you can...you know if there are any major deletions, and sequencing of the PCR products will give specific details...

...a consensus sequence is used (~90% identity between targets) or multiple target sequences are concatenated...and/or fusing your gene target sequence to a fluorescent reporter sequence (discussed below). Examples of... directly fused any gene targeting sequence to a targeting sequence for the adenine phosphoribosyltransferase...downregulation, or “silencing,” of any gene that contains sequence complementary to the dsRNA (Wilson and Doudna,...chromatin modifications of complementary gene sequences (Figure 1). As a result, gene silencing can be...fused to the DNA backbone that contains a target sequence against the fluorescent reporter in an inverted...single hairpin dsRNA is produced that contains sequence against your gene(s) of interest and the reporter...

...recognizes the DNA sequence 5´-(C/T)CCTT-3' and digests double stranded DNA at this sequence. The energy from...blue colonies that do not need to be picked and sequenced for potential positive clones. Once you introduce...restriction sites on the ends) and amplify your sequence of interest with Taq polymerase using your favorite...of your insert by PCR, restriction digest, or sequencing. Pro tips Do not add 5’ phosphates to your PCR...virus DNA topoisomerase I in Escherichia coli is sequence specific."  Proc Natl Acad Sci U S A.  1991 Nov...

...also contains a CAAX sequence to bind the mRNA to the plasma membrane; this sequence prevents diffusion ...requires inserting multiple hairpin sequences into the coding sequence and 3’ UTR. Before beginning experiments...they are bound. These regions are subsequently sequenced and aligned to the genome to determine genome-...dynamics at the single molecule level. The coding sequence of Yan et al.’s reporter contains 24 copies of...

...form a clean DNA break, Cas3 locates its target sequence and initiates single stranded DNA degradation....increase cutting efficiency and diversify PAM sequences of Cas12a to further optimize it for therapeutic...sensitive to even a single mismatch in the target sequence Similar to Cas12, it is also significantly smaller...non-complementary ssDNA when engaged by its target sequence. Why would you want to give it a go? The high ...indiscriminately cleaves ssRNA when engaged at its RNA target sequence. The RNA-targeting ability of Cas13 makes it perfect... facts – PAM requirement: protospacer flanking sequence – A, U, or C. Best for: targeting single stranded... facts – PAM requirement: protospacer flanking sequence – none. Best for: targeting single stranded RNA...

... are theoretically 256 distinct flanking sequences, sequences that differ by only one base may result ... that they cleave outside of their recognition sequence, creating four base flanking overhangs. Since ...these overhangs are not part of the recognition sequence, they can be customized to direct assembly of ...specify assembly order, so fragments with 5’ or 3’ sequence homology cannot be assembled using this method...appropriate oligonucleotides specifying a gRNA target sequence into a Cas9-containing plasmid such as pX330. ...Golden Gate cloning Golden Gate cloning is not 100% sequence-independent: to avoid undesired digestion, the...

...custom CRISPR antimicrobials can target specific sequences in a single virulent bacterial species, or even...2014, Citorik et al. and Bikard et al. developed sequence-specific, antimicrobial, plasmid-based, CRISPR... cases, they used 1-2 CRISPR crRNAs targeting sequences found only in certain bacteria (Figure 1). Citorik...targeting either genomic (blue) or plasmid-based sequences (red). Treating a mixed population, represented...targets bacteria with the given genomic or plasmid sequence from a mixed population without affecting the ...the other bacteria. Targeting a genomic sequence results in cell death. Plasmid targeting may result in ...Citorik, Robert J., Mimee, Mark, and Timothy K. Lu. “Sequence-specific antimicrobials using efficiently-delivered...

...biological process. High quality genome sequence A high-quality sequence assembly of the zebrafish genome was... will help clarify their roles in humans. The sequenced genome also permits “whole-genome/transcriptome...as the field of genetics progresses and genome sequences become available, new hypotheses about specific...organs during embryo development. Single cell sequencing-based lineage tracing Recently, gene expression... embryo has been mapped using single cell RNA sequencing approaches (Harland, 2018). Several different...such as CRISPR/Cas9, Tol2, and the high-quality sequenced zebrafish genome. One study profiled 38,731 cells...produced, rather than novel, abnormal cell types. Sequencing-based lineage tracing can help elucidate the ...

...depend more on the individual sequence(s) than on how well that sequence fits into the five rules. By ...three or four base-pair sequences, and exclude or include specific overhang sequences, allowing it to be used...specific to your DNA sequence, SplitSet is the tool for you. Once you input a sequence, SplitSet finds optimal...you’d like the tool to use to identify fusion sequences for either a circular or linear assembly. If you...even more specificity, you can exclude specific sequences or set a narrow search window for a site—or sites—that...