MiniPrep ver. 000225

 

Solution I

@@@@ 50mM glucose

@@@@ 25mM Tris-HCl@ (pH 8.0)

@@@@ 10mM EDTA (pH 8.0)

 

@@@@ Filter sterilization

 

@@@@ Cool on ice just before start mini-prep

 

Solution II

@@@@ 0.2N NaOH

@@@@ 1% SDS

 

@@@@ Fresh preparation. Keep at room temperature

 

Solution III

@@@@ 5M Potassium Acetate 60ml

@@@@ Acetic acid 11.5ml

@@@@ dH2O 28.5ml

 

TE containing RNase A

@@@@ 1ml TE

@@@@ 5µl of 10mg/ml RNase A

@@@@ or 20µg/ml RNase A


Mini Prep Procedure

l     Keep 0.5ml of bacteria cultured LB broth as a stock

l     Spin down rest of bacteria for 1min at 15000 RPM/4‹C

l     Discard the supernatant as much as possible

l     Suspend the pellet in 100µl of Solution I and mix by pipetting

l     Add 200µl of Solution II, mix well by swirling for complete lysis

l     Keep on ice for 5min

l     Add 150µl of cold Solution III and vortex well

l     Keep on ice for 5min

l     Spin for 5min at 15,000 RPM/4‹C for pelleting

l     Take supernatant (c.a. 0.5ml) and transfer to a new tube

l     Add equal volume of phenol/chloroform/isoamyl alcohol

l     Take aqueous phase to a new tube

l     Add equal volume of chloroform

l     Take aqueous phase

l     Add equal volume of isopropyl alcohol

l     Leave for 2min at room temperature

l     Spin for 10 min at 15,000 RPM/room temperature

l     Remove sup. 70% EtOH wash

l     Dry pellet briefly by speed vac.

l     Resuspend in 40µl of TE containing 20µg/ml RNase A