1) We cultured bacterial strain with the LB medium which I added ampicillin to so that density becomes 100ug/ml overnight.(We made a nutrient medium of around 5 ml in 50 ml falcons)(Against 5 ml of nutrient mediums, Amp used 5ul)
2) Aliquot 1ml culture into a 1.5 ml microcentrifuge tube,and Made it spin at 10000rpm (4℃) for 1 min to harvest the bacteria.
3) Removed supernatant and performed 2)operation again, Removed supernatant .
4) Resuspended bacterial pellet by complete vortexing in 100ul SolutionⅠ(50 mM glucose:9g, 25 mM Tris-HCl(pH 8.0):25ml, 10mM EDTA:).
5) Inverted bacterial pellet by complete fall mixtureing in 200ul Solution Ⅱ resuspension buffer,and confirmed that it became transparent.
6) Cooled for three minutes in ice.
7) Inverted bacterial pellet by complete fall mixtureing in 150ul Solution Ⅲ resuspension buffer,and confirmed that it became Cloudiness.
8) Cooled for 3 minutes in ice.
9) Harvested the DNA by spinning at 10000rpm (4℃) for 10 min.
10) Gathered only supernatant and moved it in a new microcentrifuge tube.
11) Added 0.8ul Rnase(10mg/ml) and incubate the solution(37℃,1min)
12) Added 200ul phenol:chloroform(1:1) and inverted
13) Harvested by spinning at 10000rpm (4℃) for 5 min.
14) Removed only supernatant and moved it in a new microcentrifuge tube, after that tapped in 200ul chloroform.
15) Harvested by spinning at 10000rpm (4℃) for 1 min.
16) Moved its supernatant to a new microcentrifuge tube and add 15ul 3M sodium acetate.(Don't gather underlayer)(The ratio of the 3M sodium acetate and supernatant is made to be 10:1)
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