Plasmid DNA miniprep protocol for EZ - 10 Spin Column Plasmid DNA minipreps kit ( BS413 ) , 50 preps
Add 1.5 mL of overnight culture to a microcentrifuge tube and centrifuge at 12,000rpm for 2 minutes .
Drain the clarified supernatant completely leaving only the cell pellet .
Optional : Depending on ease of plasmid replication add a further 1.5 mL of overnight culture to the microcentrifuge tube containing the previous pellet and repeat until a maximum of 5 mL of overnight culture has been added .
Add 100uL of Solution 1 to the pellet , mix well , ensuring that no clumps are present and let sit for 1 min .
Add 1μL of VisualLyse to the solution from step 2 ( optional ) .
Add 200µl of Solution II to the mixture , and mix gently by inverting the tube 4 - 6 times and then keep at room temperature for 1 minute .
To prevent contamination from genomic DNA , do not vortex .
Add 350µl of Solution III , and mix gently .
Incubate at room temperature for 1 minute .
A fluffy white material forms and lysate should become less viscous .
If VisualLyse has been added in step 3 , the suspension should be mixed until all traces of blue has gone and lysate becomes colorless .
Centrifuge at 12,000rpm for 5 minutes .
Transfer the above supernatant ( step 6 ) to the EZ - 10 column .
Centrifuge at 10,000rpm for 2 minutes .
Discard the flow - through in the tube .
Add 750µl of Wash Solution to the column , and centrifuge at 10,000rpm for 2 minutes .
Repeat wash procedure in step 8 .
Discard the flow - through in the collection tube .
Centrifuge at 10,000rpm for an additional minute to remove any residual Wash Solution .
Transfer the column to a clean 1.5ml microfuge tube ( BT620 - NS ) .
Add 50µl of Elution Buffer into the center part of the column and incubate at room temperature for 2 minutes .
Store purified DNA at - 20ºC Centrifuge at 10,000 rpm for 2 minutes
