Plasmid DNA isolation (maxi-prep)

Thursday, October 16, 2003

Description
Protocol for large scale Plasmid DNA isolation

Procedure
Y To be read as micro (mu)

1. 5ml of LB medium containing 50Yg/Yl ampicillin [Sigma A-0166] was innoculated with a single bacterial colony. The culture was incubated at 37 aC overnight with vigourous shaking (approx. 250 rpm).
2. 250ml of LB medium containing 50Yg/Yl ampicillin was innoculated with 2.5ml of the overnight culture and incubated at 37 aC overnight with vigourous shaking (approx. 250 rpm).
3. Next morning, the cells were centrifuged at 5000 x g, 4 aC for 15 minutes.
4. The pellet was resuspended in 6ml of ice-cold lysis buffer by gently pipetting cells. The suspension was incubated for 10 minutes on ice.
5. 7.5ml of freshly prepared sodium chloride [BDH 30123] / SDS solution [Sigma L-4390]. was added to the suspension and mixed by inversion only. This was incubated for 10 minutes on ice.
6. 7.5ml of ice-cold 3M sodium acetate, pH 4.6 [Qualigens 13905] was added to the suspension. It was mixed by inversion for approximately 10 seconds and incubated for 20 minutes on ice.
7. The tube was centrifuged at 12000 x g for 5 minutes.
8. The supernatant containing plasmid was transferred to a fresh tube, avoiding the white precipitate and DNase-free RNase A [Sigma R-9009] to a final concentration of 20Yg/Yl was added.
9. The solution was mixed and incubated at 37 aC for 20 minutes.
10. 0.5 volumes of tris-equilibriated phenol, pH 8.0 [SRL 1624132] and 0.5 volume of chloroform [Qualigens 12305] - isoamyl alcohol [Qualigens 21515] was added to the solution. It was vortexed for 1 minute and centrifuged at 12000 x g for 10 minutes.
11. The above extraction was repeated once.
12. The upper aqueous phase was transferred to a fresh tube and 1 volume of chloroform [Qualigens 12305] - isoamyl alcohol [Qualigens 21515] was added to it. The solution was vortexed for 1 minute and centrifuged at 12000 x g for 10 minutes.
13. The upper aqueous phase was transferred to a fresh tube and 2.5 volumes of ethanol [Merck 1.009883.0511] was added to it. It was mixed by inversion and allowed to precipitate overnight at V20 aC.
14. The DNA was recovered by centrifuging at 12000 x g for 30 minutes. The pellet was rinsed with ice-cold 70% ethanol [Merck 1.009883.0511] and air-dried.
15. The dried pellet was dissolved in 500Yl of water. Approximate yield was 500-750Yg of plasmid DNA.
16. The DNA was estimated at 260nm and 280nm on a Shimadzu UV 1601 UV-visible spectrophotometer.

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Plasmid DNA isolation (maxi-prep)