Mini-preparation (isolation & purification plasmid) ~ from : Sambrook

Isolation of plasmid :

overnite culture cell (2 ml LB)

12000 rpm, 5 min, 4oC

take pellet
+ 100 ul solution I *

vortex

+ 200 ul solution II *

swirling 6-8 x

on ice 5 min

+ 150 ul solution III *

swirling 6-8 x

on ice 5 min

12000 rpm, 10 min, 4oC

take supernatan
+ 400 ul PCI (Phenol : Chloroform : Isoamilalkohol = 25 : 24 : 1)

vortex

12000 rpm, 5 min, 4oC

take supernatant (400 ul)
+ 2.5xvolume ethanol 100% (1 ml)
+ 0.1xvolume 3M Natrium Acetate (40 ul)

mixed

incubate -20oC, 1 h

12000 rpm, 10 min, 4oC

take pellet
+ 1 ml ethanol 70% cold

12000 rpm, 10 min, 4oC

take pellet

dry up

+ 25 ul TE or H2O

Purification of plasmid :
DNA (25 ul)
+ 5 ul RNAse A (0.1 ug/ul)
+ 20 ul ddH2O

incubate 37oC, 1 h

+ (PEG : NaCl)* : DNA
3 : 5
30 ul : 50 ul

incubate 30 min, on ice

10000 rpm, 10 min, 4oC

take pellet
+ 1 ml ethanol 70% cold

10000 rpm, 10 min, 4oC

take pellet

dry up

+ 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

~ oleh adrianna pada November 29, 2009.

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