...Plasmids 101: Origin of ReplicationPlasmids 101: PCR...

...such as physically carrying out experiments like PCR and running gels, but in research you also experience...

...AAV titering by SYBR green qPCR , confirmation of the transfer plasmid with PCR, purity assessment by silver...

...vectors using BsaI, and promoter-gRNA fragments are PCR amplified for cloning into one of three Zeamays codon-optimized...

...including AAV titering by probe-based droplet digital PCR, in vitro and (when possible) in vivo testing, and...

...cell population. The region of interest is PCR amplified, PCR products are denatured-renatured, treated... The PCR product is then run on an agarose gel to determine whether the edit was successful. PCR amplification...run on an agarose gel to identify DNA fragments. PCR and restriction digest (for HDR repaired DSBs): For...novel restriction site. The region of interest is PCR amplified, digested with the appropriate restriction...run on an agarose gel to identify DNA fragments. PCR amplification and gel electrophoresis (for HDR or...deletions or insertions, the region of interest can be PCR amplified using primers that (A) flank the region...and exact sequence of targeted alleles. Involves PCR amplification of targeted region from DNA, subcloning...

...add restriction sites to any piece of DNA during PCR amplification, allowing for it to then be digested...a single adenosine (A) overhang on the 3' end of PCR products. The complementary thymine (T) comes from...an incredibly quick and easy way to clone a fresh PCR product into a plasmid. The major disadvantage of...are also available, allowing for rapid cloning of PCR products into donor plasmids without the need for...used, so if properly designed, any insert fragment (PCR product or synthesized oligo) with appropriate overhangs...add). Complementary overhangs are built into the PCR primers for the insert, based on the destination ...

...and these rare events are easily detectable using PCR. The system also functions in S. pneumoniae and can...created two “pCRISPomyces” systems for use in Streptomyces (Cobb et al., 2015). pCRISPomyces-1 includes ...array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays...pCas9: carries Cas9 and chloramphenicol resistance pCRISPR: carries a spacer targeting the gene of interest...machinery are first electroporated with pCas9. Then, pCRISPR is introduced along with an oligonucleotide repair...advantages including shorter plasmid curing time. pCRISPomyces for editing in Streptomyces  Streptomyces bacteria...

...commonly used technique for molecular cloning, such as PCR - or restriction enzyme -based cloning. Protocol:...

...annealed synthetic oligos - eliminating the need for PCR or additional molecular cloning steps during plasmid...occurred at higher frequency and could be detected by PCR while large deletions were relatively rare. The vectors...target G-protein coupled receptors (GPCRs) yet one third of the GPCRs in the human genome are orphan receptors...Mammalian Expression 74334 pCR8-Magneto2.0-p2A-mCherry Gateway 74333 pCR8-Magneto2.0 Gateway 74307...are unknown. Identifying new ligands for orphan GPCRs is of significant medical interest, however efforts...allows researchers to interrogate the druggable human GPCR-ome using a quick, affordable, and ubiquitous reporter...recruitment assay incorporating 314 codon-optimized GPCR sequences for human cell line expression. In a twist...

...(~45 minutes). Method #2 Place mixed oligos in a PCR tube. Place tube in a thermocycler programmed to ...

...commonly used technique for molecular cloning, such as PCR- or restriction enzyme-based cloning. Purifying DNA...maintaining sterility while working. AAV Titration by qPCR Use qPCR to measure the physical titer of your adeno-...adeno-associated virus preparation. AAV Titration by qPCR Using SYBR Green Technology Protocol Agarose Gel...

... This is frequently done after performing either PCR - or restriction enzyme -based cloning to test individual...

...commonly used in molecular cloning techniques, such as PCR or restriction cloning . It is also used to quickly...

...and insert DNA to use. Combine the following in a PCR or Eppendorf tube: Vector DNA Insert DNA Ligase Buffer...

...minutes at 95°C in a PCR machine or in a beaker of boiling water. If using a PCR machine, incubate the...Days post-infection mRNA knockdown (quantitative PCR) ≥3 days Protein knockdown (western blot) ≥4 days...

... onto the insert of interest using PCR. This is accomplished by PCR amplifying the insert sequence using...restriction sites onto the insert of interest using PCR. The process of subcloning consists of digesting ...

...restriction sites to flank your insert : You can use PCR Based Cloning and add restriction sites to the ends...

...surviving cells are collected, their plasmids are PCR-amplified and sequenced using NGS to identify their...

...controlling GPCR activation have been developed to selectively study individual types of GPCRs with greater...be used for lower frequency stimulation. GPCR signaling GPCR signaling is a complicated process involving...change the membrane potential. OptoXRs are modified GPCRs. When exposed to light, they activate downstream... coupled receptors  G-protein coupled receptors (GPCR) regulate cellular function through multiple intra-cellular...have been developed for utilizing light to control GPCR activation and downstream signaling cascades (reviewed...Levitz, 2020), including light-sensitive chimeric GPCRS (OptoXRs) (Tichy et al., 2019), photoswitchable ...rhodopsin, and the intracellular domain of an endogenous GPCR, such as adrenergic receptors. When exposed to light...