...plasmid, excluding any restriction enzymes or vector purchases (Table 1). Restriction enzymes and vector ...also a great way to get to know people doing interesting and relevant work in your field! However, there...work done previously, borrow small aliquots of restriction enzymes for one-off projects, or even save yourself...

...your cloning strategy: Restriction enzyme digestion Be mindful of the restriction enzymes you chose. Make...which technique you are going to adopt (i.e. restriction cloning, Gibson Assembly, Golden Gate etc.), ... fragments of the size you expect, check the restriction pattern of the enzymes you chose, and verify ...

...iterative one-pot reactions with straightforward restriction digestion and ligation steps to build barcoded...flexible and can accommodate any genetic elements of interest. It can thus be tailored to address the users’...effective gRNA sequences targeting your genes of interest. Thanks to the tremendous efforts made by various...the University of Hong Kong. He is particularly interested in developing and applying new technologies to...

...edition, we’re going to take a look at a very interesting tool that can be used for creating (excuse the...-lox” or “LSL” cassette) upstream of a gene of interest will prevent gene expression in the absence of...described above, a genetic modification can be restricted based on experimental constraints. This has been...used to successfully introduce Cre into cells of interest. What if I need two separate recombination events...

...vector file, and quickly clone their vector of interest. And it truly is plug-and-play. Because of its...recommend checking your cloning via a miniprep and restriction digest, and then plasmid sequencing if you can...Cloning specifics Fragmid uses Bbsl, a Type II restriction enzyme that cleaves outside of its recognition..., 93% had the correct banding pattern in the restriction enzyme digest. They sent 82 of the correctly ...

...inserting a targeting vector containing the gene of interest into a zygote, or into embryonic stem cells that...gene construct can be used to insert the gene of interest into a zygote or into embryonic stem cells. Non-homologous...genome using homology arms that flank the gene of interest. In either case, a positive selection marker such...the genome with similar homology to the site of interest or create mosaic animals where not all cells in...

...promoters could be an easier way to restrict expression of a gene interest in an animal model. A toolkit ...Applications for synthetic promoter AAVs Besides restricting AAV transgene expression to particular cell types...types, pairs of synthetic promoter can be used to restrict expression to a single cell type based on the ...

...finding journal articles related to your actual interests while also staying on top of the ever expanding...which is a nice way to see growth or decline of interest in a field over time. Creating an alert for a ...places to look for more articles on your topic of interest.  Best uses Accessing the vast body of biomedical...keywords, concepts, journals, and/or papers of interest. You can also just follow feeds curated by Meta...

...transduced cells and loss of transgene expression (Interestingly, the strong immunogenicity of AdVs is what makes... in trans. The Gutless AdV have generated high interest for gene therapy due their increased cloning capacity...plasmids: a shuttle vector (in which the transgene of interest is cloned) and pAdEasy™ which contains the adenoviral...plasmid stock should be analyzed by endonuclease restriction digest or fully sequenced to confirm its integrity...

...since 2015 to conditionally degrade proteins of interest in cells (Zhang et al., 2015). Recently the Ward...degron (AID) sequence attached to your protein of interest. TIR1 interacts with endogenous Skp1 and Cul3 ...) tagged proteins. Degradation of your protein interest would also result in depletion of the FP, which...integrate the tagged AID* into genomic sites of interests via CRISPR techniques. This tool-kit includes ...

... based off of p24 quantification tend to be overestimates since they can include free p24 and defective...assay, titering via measurement of viral RNA can overestimate the amount of infectious virus in a prep as ...physical titer, there is a risk that they may underestimate viral titer as these assays cannot distinguish...integration events, reducing the possibility of underestimating the viral titer. The main drawback of this ...

...select for sybodies targeting a membrane protein of interest using the Sybody Generation Toolbox and commercially... (for purification purposes) protein target of interest, the protocol goes through 3 major steps to select...exchange (FX) cloning technique which uses Type IIS restriction sites (Geertsma, 2013). Once the phagemids are...bind strongly and specifically to the protein of interest. The Seeger lab used the ELISA technique described...

...transcribe, translate and modify your protein of interest. Modified proteins can then be extracted from ...is quite simple; DNA coding for your protein of interest is inserted into a plasmid expression vector that...propagate, are induced to produce your protein of interest, and then lysed. Protein can then be purified ...from Northeastern University and is particularly interested in cell signaling and communication. She loves...

...allowing the reference sequence and the gene of interest to be amplified in the same reaction. Taqman’s...contains a primer and probe set for both the gene of interest and the reference sequence. At the end of the ...the number of positive droplets for the gene of interest and reference gene, copy number can be determined... your experiment?  Complexity of the gene of interest Some gene sequences are harder to amplify and...

... (5’) and downstream (3’) of the DNA region of interest. Primers are crucial for this process as DNA polymerases...times to exponentially replicate the target DNA of interest. This entire reaction used to be done manually...used in molecular cloning to create a plasmid of interest, for detecting the presence of an insert or piece...End point PCR or just PCR Amplify a region of interest  Molecular Cloning Site-directed mutagenesis...

...adjacent to the targeted sequence at the locus of interest. SpCas9’s 3’ NGG PAM occurs frequently in GC-rich...progress we’ve made, there is still a need for and interest in enzymes with shorter PAM sequences and/or broader...permits quick, large-scale mutation of the gene of interest. They created a selection phage (SP) to carry ...-3.13. To test xCas9 3.0-3.13, Hu et al. first restored catalytic residues D10 and H840. To ensure that...

...is to replace the genomic copy of your gene of interest with a fluorescent protein fusion. CRISPR genome...scientists to tag and light up endogenous genes of interest to better understand normal protein function. ...vectors:  1) a gRNA vector that targets the gene of interest, 2) a plasmid to specify the reading frame of ...flanked by long regions of homology to the gene of interest to promote homology directed repair after a CRISPR...

...that directs them to the promoter of a gene of interest, they have been shown to dramatically alter levels... transcriptional start site (TSS) of a gene of interest, it induces potent gene expression with no genetic...of the transcriptional start site of a gene of interest, Cas9 forms a steric block that prevents RNA polymerase...the length of gRNAs targeting different sites of interest thus represents a straightforward means of exerting...

...You're OK. However, if you fuse your protein of interest (POI) to GFP to study the protein's behavior in...reticulum (OSER) whorls. 2)A dimerizing protein of interest fused to a dimerizing FP has potential to form...protein concentrations. An imperfect solution is to restrict imaging analysis to cells expressing the lowest...Department of Anatomy and Structural Biology. His interests include the quality control of secretory proteins...

...initial reversal drive cannot restore the exact original sequence, it can restore the original protein-coding...using a recoding strategy; a subsequent drive can restore the wild-type sequence (save for the residual sgRNAs...immunizes it against the first drive. Laboratories interested in building candidate gene drives intended for...