WIZARD PLUS MINI-PREP
 
Last Update: December 2006
 
Based on protocol from: Promega
 
PREPARATION OF E. COLI
1. Use a single colony from a LB agar plate (containing antibiotic) to inoculate 10 mL of LB medium (containing the same antibiotic)
2. Incubate over night (12 to 16 hours) at 37°C
   
PRODUCTION OF CLEAR LYSATE
1. Harvest 1 to 5 mL (high copy number plasmid) or 10 mL (low copy number plasmid) of bacterial culture by centrifugation for 5 mins at 10,000 x g in a table-top centrifuge. Pour off the supernatant and blot the inverted tube on a paper towel to remove excess media
2. Add 250 mL of Cell Resuspension Solution and completely resuspend the cell pellet by vortexing well or pipetting. It is essential to thoroughly resuspend the cells. If not already in a microcentrifuge tube, transfer the resuspended cells to sterile 1.5 mL microcentrifuge tube(s)
3. Add 250 mL of Cell Lysis Solution and mix by inverting the tube 4 times (do not vortex). Incubate until the cell suspension clears, approximately 1 to 5 mins. Do not incubate for longer than 5 mins

4. Add 10 mL of Alkaline Protease Solution and mix by inverting the tube 4 times. Incubate for 5 minutes at room temperature. Alkaline protease inactivates endonucleases and other proteins released during the lysis of the bacterial cells that can adversely affect the quality of the isolated DNA
5. Add 350 mL of Wizard® Plus SV Neutralization Solution and immediately mix by inverting the tube 4 times (do not vortex)
6. Centrifuge the bacterial lysate at 14,000 xg in a microcentrifuge for 10 mins at room temperature
   
cDNA ISOLATION
1. Transfer the cleared lysate (approximately 850 mL) to the prepared Spin Column by decanting. Avoid disturbing or transferring any of the white precipitate with the supernatant
2. Centrifuge the supernatant at 14,000 xg in a microcentrifuge for 1 min at room temperature. Remove the Spin Column from the tube and discard the flowthrough from the Collection Tube. Reinsert the Spin Column into the Collection Tube
3. Add 750 mL of Column Wash Solution, previously diluted with 95% ethanol, to the Spin Column
4. Centrifuge at 14,000 xg in a microcentrifuge for 1 min at room temperature. Remove the Spin Column from the tube and discard the flowthrough. Reinsert the Spin Column into the Collection Tube
5. Repeat the wash procedure using 250 mL of Column Wash Solution.
6. Centrifuge at 14,000 xg in a microcentrifuge for 2 mins at room temperature.

7. Transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube being careful not to transfer any of the Column Wash Solution with the Spin Column. If the Spin Column has Column Wash Solution associated with it, centrifuge again for 1 min at 14,000 xg before transferring to the new 1.5ml tube
8. Elute the plasmid DNA by adding 100 mL of Nuclease-Free Water to the Spin Column. Centrifuge at 14,000 xg for 1 min at room temperature in a microcentrifuge.
9. After eluting the DNA, remove the assembly from the 1.5ml microcentrifuge tube and discard the Spin Column
10. DNA is stable in water without addition of a buffer if stored at &endash;20°C or below. DNA is stable at 4°C in TE buffer. To store the DNA in TE buffer, add 10µl of 10X TE buffer to the 100µl of eluted DNA

11. Cap the microcentrifuge tube and store the purified plasmid DNA at -20°C or below