ReadiPrep™ Plasmid Preparation Mini Kit

1. Resuspend the bacterial pellet in resuspension buffer.
2. Add equal volume of lysis buffer, mix gently, followed by addition of neutralization buffer and centrifuge to pellet out the debris.
3. Load the supernatant onto the column, spin and discard the flow through.
4. Wash the column with binding buffer followed by wash buffer.
5. Elute the plasmid in elution buffer.

Prepare ReadiPrep™ Wash Buffer (WB1) working solution by adding 10 mL of Ethanol (96-100%, not provided) into the ReadiPrep™ Wash Buffer (WB1) (Component G). 

Prepare ReadiPrep™ Resuspension Buffer (RB1) working solution by adding whole vial of RNAse (Component B) into the ReadiPrep™ Resuspension Buffer (RB1) (Component A).
Note: After addition of RNAse, ReadiPrep™ Resuspension Buffer (RB1) working solution must be stored at 4 ℃.

The protocol is designed for the purification of plasmid DNA from 1 to 5 mL of overnight culture.

1. Resuspend the bacterial pellet in 250 µL of ReadiPrep™ Resuspension Buffer (RB1) working solution.
2. Add 250 µL of ReadiPrep™ Lysis Buffer (LB1) (Component D) and mix thoroughly by inverting the tube 5-6 times.
   Note: Do not vortex. Do not allow the lysis reaction to proceed for more than 5 minutes.
3. Add 350 µL of ReadiPrep™ Neutralization Buffer (NB1) (Component E) and mix immediately and thoroughly by inverting the tube 4-6 times.
4. Centrifuge for 10 mins at 13,000 rpm in a table-top centrifuge.
5. Add 800 µL of the supernatant to the ReadiPrep™ Spin Column (Component C). Centrifuge at 13,000 rpm at room temperature for 1 min. Discard the flow-through.
   
6. Add 800 µL of the supernatant to the ReadiPrep™ Spin Column (Component C). Centrifuge at 13,000 rpm at room temperature for 1 min. Discard the flow-through.
7. Wash the column by adding 750 µL of ReadiPrep™ Washing Buffer working solution (WB1). Centrifuge at 13,000 rpm at room temperature for 1 min.  Discard the flow-through.
8. Perform one more round of centrifuge at 13,000 rpm at room temperature for 1 min to remove the residual wash buffer from the columns.
9. Place the column in a clean 1.5 mL micro centrifuge tube (Not provided). Add 50 µL of ReadiPrep™ Elution Buffer (EB1) to the center of the column, incubate for 1 min and centrifuge at 13,000 rpm for 1 min.