Mini-preparation (isolation & purification plasmid) ~ from : Sambrook
Isolation of plasmid :
overnite culture cell (2 ml LB)
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12000 rpm, 5 min, 4oC
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take pellet
+ 100 ul solution I *
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vortex
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+ 200 ul solution II *
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swirling 6-8 x
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on ice 5 min
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+ 150 ul solution III *
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swirling 6-8 x
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on ice 5 min
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12000 rpm, 10 min, 4oC
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take supernatan
+ 400 ul PCI (Phenol : Chloroform : Isoamilalkohol = 25 : 24 : 1)
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vortex
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12000 rpm, 5 min, 4oC
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take supernatant (400 ul)
+ 2.5xvolume ethanol 100% (1 ml)
+ 0.1xvolume 3M Natrium Acetate (40 ul)
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mixed
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incubate -20oC, 1 h
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12000 rpm, 10 min, 4oC
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take pellet
+ 1 ml ethanol 70% cold
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12000 rpm, 10 min, 4oC
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take pellet
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dry up
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+ 25 ul TE or H2O
Purification of plasmid :
DNA (25 ul)
+ 5 ul RNAse A (0.1 ug/ul)
+ 20 ul ddH2O
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incubate 37oC, 1 h
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+ (PEG : NaCl)* : DNA
3 : 5
30 ul : 50 ul
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incubate 30 min, on ice
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10000 rpm, 10 min, 4oC
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take pellet
+ 1 ml ethanol 70% cold
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10000 rpm, 10 min, 4oC
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take pellet
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dry up
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+ 20 ul TE
* Solution I : 50 mM glucose ; 25 mM Tris-Cl (pH.8) ; 10 mM EDTA (pH.8) ; autoclaved and keep on -20 degree celcius
* Solution II (make freshly) : 0.2 N NaOH ; 1% SDS
* Solution III : 5 M Potassium Acetate (60 ml) ; glacial acetic (11.5 ml) ; H2O (28.5 ml)
* PEG : NaCl : 20% PEG 6000 ; 2.5 M NaCl ; autoclaved
