Plasmid Purification

Plasmid Miniprep Kit

Before Starting:

1. Grab Ice Bucket. Place ZymoPURE P3 on ice for 30 minutes before using.

2. Prepare Buffer:

a. Make 95% EtOH. For 50mL, add 47.5mL 100% EtOH to 2.5mL Mili-Q H2O.

b. Add 42mL of this to 11mL of ZymoPure Wash2 Concentrate (D4209).

3. The ZymoPURE P2 and ZumoPURE Binding Buffer may have precipitated. a. IF SO-dissolve the precipitate by incubating the bottles at 30-37 degrees C for 10-20 minutes and mix by inversion. DON’T microwave.

Starting: (Perform everything at room temperature (15-30 degrees C). 1. Obtain flasks with cloudy bacteria solution. Should be 10mL per antibiotic. Split each flask between two 1.5mL tubes.

a. Centrifuge 5mL of bacterial culture in a clear 1.5mL tube at full speed for 20 seconds in a microcentrifuge. Discard the supernatant. Gradually add volume of culture until you get through 5mL of the culture, per tube.

2. Add 250uL of P1 (Red-should be in fridge) to the pellet and resuspend cells but pipetting and vortexing.

3. Add 250uL of P2 (Green) and IMMEDIATELY MIX by gently inverting the tube 6-8 times. DO NOT VORTEX! Let sit at room temp for 3 mins. This addition lyses the cells, and the solution should be clear, purple, homogenous and viscous when the process is done.

4. Add 250uL of ice cold P3 (Yellow) and mix throughout by inverting. DO NOT VORTEX. Invert the tube again 3-4 more times after the sample turns completely yellow. It will turn yellow when the neutralization is complete and a yellowish precipitate will form.

5. Incubate the neutralized lysate on ice for 5 minutes.

6. Centrifuge this neutralized lysate for 5 minutes at 16,000 x g.

7. Transfer 600uL of the supernatant from step 6 into a clean 1.5mL microcentrifuge tube. Be careful not to disturb the yellow pellet and avoid transferring any cellular debris to the new tube.

8. Add 275 uL of ZymoPURE Binding Buffer to the cleared lysate from step 7 and mix thoroughly by inverting the capped tube 8 times.

9. Place a Zymo-spin II-P column in a Collection Tube and transfer the entire mixture from step 8 into the Zymo-Spin II-P Column.

10. Incubate the Zymo-Spin II-P/Collection Tube assembly at room temperature for 2 minutes and then centrifuge at 5,000 x g for 1 minute Discard the flow through

11. Add 800 uL of ZymoPURE Wash 1 to the Zymo-Spin II-P Column and centrifuge at 5,000 x g for the 1 min. Discard the flow through.

12. Add 800 uL of ZymoPURE Wash2 to the Zymo-Spin II-P Column and centrifuge at 5,000 x g for 1 min. Discard the flow though. Repeat this wash step with 200 uL of ZymoPURE Wash 2.

13. Place the Zymo-Spin II-P Column in a Collection Tube and transfer to a microcentrifuge. Centrifuge at > or at 10,000 x g for 1 minute in order to remove any residual wash buffer.

14. Transfer the Zymo-Spin II-P Column into a clean 1.5 mL tube and add 25uL of ZymoPURE Elution Buffer directly to column matrix. Incubate at room temperature for 2 minutes and then centrifuge at over or equal to 10,000 x g for 1 minute in a microcentrifuge.