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/4C
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/4C 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