Editing Plasmid MaxiPrep Aqueous Two Phase System

Alkaline Lysis<br/>Cells (250 mL) were harvested by centrifugation at 15.000x g (20 min. 4 0&nbsp;C) and the&nbsp;pellets suspended in 8 ml of 50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA, pH: 8 (buffer 1)&nbsp;<br/>The cells were lysed by adding and gently mixing (10 min in ice) 8ml of 200 mM NaOH, 1%&nbsp;(w/v) sodium dodecyl sulfate (buffer 2). The lysate was neutralized with 8 ml of a solution of 3M&nbsp;potassium acetate, 11.5&nbsp;% (v/v) glacial acetic acid (10 min on ice) (buffer 3). All buffers were<br/>previously chilled. After neutralization, cell debris, protein and gDNA precipitates were removed&nbsp;by centrifugation (15.000 x g, 20 min. 4 0<br/>C). The second centrifugation at 15.000 g, 20 min. at the&nbsp;same temperature was performed to further clarify the supernatant. The plasmid containing lysate&nbsp;is precipitated by 0.6 vol of isopropanol at 4 oC.



Solublize DNA using 2~&nbsp;2.4 M ammonium sulphate, inclubate at 4 oC, spin again to remove insoluble aggregate.



Aqueous two - phase Extraction&nbsp;

ATPS were prepared in 15 ml graduated tube with conical tips by adding distilled water,&nbsp;ammonium sulphate, PEG 600, Escherichia coli lysate and, up to a total weight of 5 g. The&nbsp;amount of lysate loaded was 1 g [12]. The components were added in the following order; PEG,&nbsp;salt, water, lysate. After thoroughly mixing, the mixture was centrifuged for 30 min.15000 g at&nbsp;4 oC. Top and bottom phase were isolated and stored at 4oC for analysis. &nbsp;

PEG 600 35&nbsp;% (w/w) - (NH4)2SO4 6% (w/w). 20&nbsp;% (w/w) lysate

The bottom phase contains plasmid DNA.

To precipitate DNA in the bottom phase, add&nbsp;