Isolating low-copy plasmid DNA

Isolating low-copy plasmid DNA

Abstract Plasmids differ significantly in their copy number due to their origin of replication, size, and associated insert. A number of plasmids also contain mutations that allow them to reach very high copy numbers within the bacterial cell. However, depending on the objectives of the experiment or on the nature of the cloned insert, it is not always possible to use high-copy number plasmid DNA. Preparing low-copy number plasmid DNA can become a significant burden for applications that require large quantities of highly concentrated endotoxin-free plasmid DNA, such as transfections and in vivo genetic manipulations. In these cases, it requires either scaling up the prep, performing multiple preps, and/or concentrating the plasmid following of purification. Zymo Research has developed an innovative protocol for the ZymoPURE™ Maxiprep Kit that enables purification of high quantities of plasmid DNA from low copy number plasmids. Using this protocol, researchers are able to quickly and efficiently isolate twice as much transfection grade low-copy number plasmid DNA at workable concentrations in 20 minutes using a microcentrifuge column when compared with standard procedures.

 

To learn about isolating DNA from any samples, including brain, see the Quick-DNA™ Plus Kit.

 

herpes simplex virus type-1, HSV-1

Figure 1. Latent HSV-1 DNA has been found in CNS neurons with asymptomatic reactivations possibly leading to early neurodegeneration.

 

Leyton et al. (2015) evaluated known natural activators of the AMPK/Sirt 1 sensors involved in neuronal survival pathways and neuroprotection, including Resveratrol and Quercetin, to determine if activation could reduce HSV-1 propagation or counteract the effects of CNS neuronal infection. The Quick-DNA™ Plus Kit was utilized for isolation of DNA from HT22 neuronal cells for infectivity studies. Results of the study suggested that activators of the AMPK/Sirt 1 axis increased the viability of infected neurons, reduced viral titer in the supernatant, and reduced the expression of viral genes. Pretreatment with Resveratrol and Quercetin reduced the cleaving and hyperphosphorylation of tau that is normally associated with HSV-1 infection. This indicates that Resveratrol and Quercetin could be useful in reducing the risk of HSV-1 infection in neurons and the cellular damage caused by reactivation events that have been associated with early neurodegeneration, such as in Alzheimer’s disease.

 

Citation
Leyton, L. et al. (2015) Nutraceutical activators of AMPK/Sirt1 axis inhibit viral production and protect neurons from neurodegenerative events triggered during HSV-1 infection. Virus Research.



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