Paul.xu at 19:13, 29 July 2016

2016-07-29T19:13:38Z

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

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

Aqueous two - phase Extraction 

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

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

The bottom phase contains plasmid DNA.

To precipitate DNA in the bottom phase, add 

Plasmid MaxiPrep Aqueous Two Phase System - Revision history

Paul.xu at 19:13, 29 July 2016