کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5136541 | 1494016 | 2017 | 8 صفحه PDF | دانلود رایگان |
- Partitioning of pDNA in ATPSs containing (PEGÂ +Â Na2SO4) was studied.
- Partitioning of proteins in the lysate was studied in aqueous (PEGÂ +Â Na2SO4) system.
- Affinity partitioning of pDNA using a TFO as an affinity ligand was investigated.
Extraction of plasmid DNA (pDNA) from an alkaline bacterial cell lysate has been investigated using partitioning and affinity partitioning in aqueous two-phase systems (ATPSs) containing 12% (w/w) polyethylene glycol and 12% (w/w) sodium sulfate. In this work, a modified 20Â bp pyrimidine oligonucleotide acting as a triplex-forming oligonucleotide (TFO) was employed as an affinity ligand to improve the partition behavior. The pUC118 plasmid was chosen to be extracted because of its specific oligonucleotide recognition sequence. In order to find the proper conditions, operational parameters such as PEG molecular weight, pH and lysate load were also studied. The results showed that adding the affinity ligand strongly changed the partitioning behavior of pDNA without any considerable impact on the partitioning of RNA and protein content. The best achieved result was at pHÂ =Â 6 and PEG MW of 600Â Da and 60% of lysate load. In these conditions, 67% of pDNA was recovered in the top phase while the contaminants mainly accumulated in the bottom phase.
Journal: Journal of Chromatography B - Volumes 1044â1045, 15 February 2017, Pages 112-119