...with a 1.9 kb stuffer sequence that can be removed and replaced with an shRNA sequence of your choice. More...libraries and can be combined with high-throughput sequencing to perform multiplexed analysis of cells pre-...backbones) here. Cloning and expressing new shRNA sequences pLKO (Plasmid #10878) This plasmid has been used...

...Yamagata et al used previously published nanobody sequences that targeted the following antigens: GFP, RFP...actin-binding protein gelsolin, but any nanobody sequence could be used to make a RANbody. RANbodies are...secreted from cells due to their IgGκ signal peptide sequence, making them easy to harvest from cell culture...useful RANbody, consider leaving a link to its sequence in the comments below. References Sanes, J.R....

...phosphate backbone modifications The recognition sequence for CRISPR-Cas9 targeting is led by a ~20 bp gRNA...CRISPR RNA (crRNA). In addition to the 20 bp guide sequence, the crRNA also contains a ~20 bp repeat region...validated gRNA repository to find pre-tested gRNA sequences for your next CRISPR experiment.    Chemical...which is photocaged and unable to engage a target sequence. Upon a brief light exposure (several seconds)...

...easy enough to contemplate DNA in terms of its sequencing, in vivo studies should always take DNA’s chromatin...expression pattern and an auxin inducible degron (AID) sequence attached to your protein of interest. TIR1 interacts...of auxin, this TIR1 complex binds to the degron sequence ultimately leading to the degradation of the AID-tagged...(minimal 44 amino acids auxin inducible degron sequence) tagged proteins. Degradation of your protein ...

...adding long-read sequencing capabilities to complement our next-generation sequencing, setting new standards... We’re also building a portal to easily share sequences and characterization data for over 150,000 plasmids...

...containing the modifying sequence flanked by approximately 50 base pairs of sequence homologous to the chromosomal... typically contain both a protein modification sequence and a selectable gene product for isolation of...

... reaction for traditional restriction cloning. Sequence and Ligation Independent Cloning (SLIC) could ...Invitrogen and based on lambda phage integration sequences, relies on site-specific recombination reactions...components and define their final order through sequence homology. Similarly, Golden Gate assembly allows...FastCloning: a highly simplified, purification-free, sequence-and ligation-independent PCR cloning method." ...

... the exact original sequence, it can restore the original protein-coding sequence using a recoding strategy...synthetic sequence will only spread in transgenic laboratory populations with that sequence2. Both methods...; a subsequent drive can restore the wild-type sequence (save for the residual sgRNAs and possibly cas9...unpredictable ecological effects. Because the consequences of such a mistake would necessarily extend far...

...immunoprecipitation followed by high-throughput sequencing, ChIP-Seq, is the go-to method for mapping where...noise, reducing the read depth necessary when sequencing and lowering the amount of tissue needed per ...subsequent library preparation and high-throughput sequencing. Improvements to the CUT&RUN method and using...

...Transposons are sequences of DNA that can move around in a genome. In a laboratory setting, transposons...the tranposon to integrate. Additionally, the TA sequence is duplicated near the gene of interest insertion...between a greater number of extinct transposon sequences and then testing various combinations of changes...

...?  scFv sequences have traditionally been cloned by amplifying the VLand VH antibody sequences of hybridomas...semi-synthetic or entirely synthetic VL and VH sequences. Phage display also allows for screening scFv ...antibodies. To do this, antibody variable domain sequences are expressed as scFvs and screened with in vitro...assays to select for strong binders. Then the sequences for the variable domains of the selected scFvs...

... generating insertions or deletions in the DNA sequence. This same approach is effective to target short...very small deletions and insertions in the DNA sequence can completely disrupt expression of the transcribed...Cas9 generates double-strand breaks, a new DNA sequence can be knocked-in, or introduced, into the break...off-target effects since promoter and enhancer sequences remain intact (Engreitz et al. 2019). The interactions...

...The raw data is then normalized to a reference sequence present in the same amount in all samples (usually...to run multiplex assays, allowing the reference sequence and the gene of interest to be amplified in the...for both the gene of interest and the reference sequence. At the end of the run, each droplet is read as...Complexity of the gene of interest Some gene sequences are harder to amplify and quantify than others...

... editing. PAM sequences can be from 2-6 base pairs in length and follow the DNA sequence targeted by the...complex, promoting its interaction with the degron sequence and ultimately its degradation. While other eukaryotes...

...use a plasmid if it didn’t include any plasmid sequence data? Of course not. Unfortunately, the vast majority... majority of antibodies are supplied with no sequence information, leading to problems with reproducibility...affinity reagent screening, and next-generation sequencing of hybridomas have opened the door for a new ...

...sgRNA) that bears complementarity to a 20bp target sequence (Qi et al. 2013). Modifying the CRISPRi system... will bind to a PAM-adjacent target nucleotide sequence then subsequently recruit a transcriptional regulator...Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152...

...please contact us. Full sequencing of the Viral Genome Next-generation sequencing is performed on viral ...genes, and a plasmid encoding adenoviral helper sequences. AAV preparations are purified by iodixanol gradient...genomes isolated from the final AAV preparation. Sequencing results are analyzed to confirm the identity ...

... a genetic sequence and integrate it into any DNA sequence containing the 38-50 bp sequence recognized...they optimized the specific attachment sites via sequence engineering, enabling even more efficient integration...

... effective, ivTRT may be more challenging for sequences with complex secondary structure. Today, multiple.... Miura et al. (2018) suggest using a 50-80 bp sequence around the desired insertion site to search for...C-terminal insertion with Easi-CRISPR. Potential guide sequences (arrows) and their PAM (bold highlighted text)... knock-in design may naturally remove the gRNA sequence or place it far from the PAM; if not, you can ...guide. This design also separates the gRNA target sequences from their PAMs, preventing re-cutting after editing...

... a ~20 nucleotide targeting sequence and ~75 nucleotide scaffold sequence) is consistent between plants...contain a plant pol III promoter and gRNA scaffold sequence and allow researchers to insert targeting oligos...is commonly tagged with a nuclear localization sequence to enhance targeting to the nucleus, and several...the specifics - promoters used, precise protein sequences or domains, and methods of delivery - the underlying...