We narrowed to 842 results for: Eras

... comes from using Taq polymerase for the amplification step since Taq polymerase leaves a single deoxyadenosine...in the pair comes from a topoisomerase I-linearized backbone. DNA topoisomerase I (depicted as a green ...covalently linked to topoisomerase. Vectors in these kits also often have the topoisomerase site inserted into...mind that Taq polymerase has an error rate of about 1 in 3,500 bases. Typically polymerases with proofreading...Topoisomerase based cloning (TOPO cloning) is a DNA cloning method that does not use restriction enzymes...technique specifically uses Vaccinia virus-isolated topoisomerase I as this enzyme recognizes the DNA sequence...cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase I (1). If a 5′ hydroxyl group from a different...

...cell type coverage in rodent PNS AAV-PHP.S effectively transduces neurons in the peripheral nervous system... definition of systemic capsids. After some consideration, we offer this definition: A systemic capsid...CNS), crossing the BBB, and those targeting the peripheral nervous system (PNS). (Capsids, if you recall...capsids are engineered, as well as highlight some considerations for their use. Then, we’ll provide information...health. While it’s always helpful to start with a literature search to see what others have done, you’ll need...- or region-specific transgene expression. (a) Several engineered capsids with unique tropisms are now...IV injections are typically administered in the lateral tail vein or retro-orbital sinus. IT injections...

...diverse libraries of genetic variants. Several methods for generating mutant libraries exist, but none provide...per nucleotide per generation vs. 10 x 10-10 mutations per nucleotide per generation of wild-type E. coli... Lab at Berkeley, provides a new approach for generating novel genetic variants in bacteria. Read on to...applications. Challenges of existing mutagenesis methods Several continuous diversification methods exist, but there...growth conditions. Expression of an error prone polymerase I (PolI3M) is capable of mutagenesis in E. Coli...with error prone PCR, EvolvR uses an error prone polymerase to introduce mutations. However, by using nCas9... Figure 1: EvolvR consists of an error-prone polymerase (PolI) that is fused to a nicking Cas9. A guide...

... own RNA-dependent RNA polymerase into the infected cells. The EBOV polymerase replicates the viral genome...transcribe the reporter gene (e.g. firefly luciferase). The polymerase recognizes the gene start signal and ...viral infection), producing firefly luciferase. Firefly luciferase can readily be measured to quantify...under control of a T7 RNA polymerase promoter sequence. Thus, T7 RNA polymerase also needs to be present...single (non-viral) reporter gene (e.g. firefly luciferase or eGFP) flanked by the EBOV-specific gene start...replication and reporter gene transcription by the EBOV polymerase (reviewed in 2, 5). The minigenome is capable...nucleoprotein, NP. NP associates with the viral polymerase, which is formed by a complex of two proteins...

...mice to generate F1 Once you’ve performed your first cross, congratulations! You’ve generated F1 mice ...at the other. 3. Breed your F1 generation Now, your goal is to generate mice that are homozygous for all...progeny from future generations. 4. Continue breeding your colony You’ll want to generate enough mice that...there are many considerations for effectively breeding your mice. Because their generation involves the ... parental generation, they are likely to stay on separate chromosomes in future generations as well, a...modifications into mouse embryos. But once you generate a growing colony of genetically engineered mice...genotype at every allele decreases. Crossing mice to generate a desired phenotype Instead, genetic engineering...

...single RNA polymerase for transcription, eukaryotes have three RNA polymerases (Polymerases I, II, and...different types of RNA: Polymerase I is responsible for ribosomal RNA, Polymerase II is responsible for ...noted that each eukaryotic RNA polymerase terminates differently. Polymerase III, for example, relies on ...dependent, Polymerases I and II employ different mechanisms to terminate transcription. Polymerase I uses ...mechanism, whereas Polymerase II termination is more complex and involves two RNA polymerase-associated proteins...to express genes in a given host cell type are generally broken down into two broad categories, prokaryotic...polyadenylation vary across cell types, but polyadenylation generally serves to promote transcript longevity in eukaryotes...

...this. Plasmid library generation prior to one pot saturation mutagenesis Several methods can be used to...mutant strand: A mix of degenerate primers and the high-fidelity Phusion polymerase are used to introduce...interest’s function. This technique relies on the generation of a plasmid library that expresses all desired...selection. Key to this process is the ability to generate full libraries of mutant proteins. Researchers...a quick and easy technique that can be used to generate complex libraries of mutant plasmids ready for...libraries. While each of these methods works well to generate libraries, they all have inherent shortcomings...with a “sloppy” version of PCR that results in a polymerase error rate of up to 2% per nucleotide position...

...Both components of the chimera are encoded by DNA. Since researchers can generate almost any DNA sequence...whether the chimera is still functional. Final words on fluorescent protein fusions There are several choices...proteins is relatively straightforward. However, generating a fusion while keeping all of the native properties...challenging. In this blog I discuss strategies to generate fusion proteins and highlight some aspects of ...“stickiness.” Since the fluorescent tag should operate as an inert light emitting module, any tendency...the number of binding sites per unit.  There are several ways to measure stickiness. In vitro, ultracentrifugation...in cells. In addition to measuring stickiness, several researchers have also collected data on fluorescent...

...Renilla luciferase variant fused to an FP; when supplied with a luciferase substrate, the luciferase transfers...synthesized diacetyl coelenterazine-h. Unlike coelenterazine-h, diacetyl coelenterazine-h does not autooxidize...the media. Cellular esterase then converts diacetyl coelenterazine-h to coelenterazine-h, producing a constant...Renilla luciferase has a low quantum yield, and when transiently expressed in cells, generates much less...excitation light source. Instead, a luciferase substrate like coelenterazine is supplied in the culture medium...Nano-lantern, a fusion of a mutagenized Renilla luciferase (RLuc8) with Venus. This luminescent technology...transfer (BRET). Photons emitted by a Renilla luciferase variant are used to excite the fused fluorescent...

...diagnostic tools and therapeutics. In addition, the strategic use of whole phages as therapeutics, which is also...downstream manipulations. Phi29 polymerase is a phage-derived polymerase that can perform whole genome ...for 1 h at room temperature. After this, it is helpful to subject your membrane to several freeze/thaw cycles...around 8-12 h at room temperature as opposed to overnight at optimal growth temperature. If you’re lucky enough...however, a physical fragmentation strategy is preferable. Nebulization involves forcing DNA through a ...fragmentation, repair sheared ends with T4 DNA polymerase (to create blunt ends) and ligate into a bluntly...manipulation, amplifying phage DNA with phi29 polymerase prior to fragmentation can greatly facilitate...