Cong T. Trinh:mini prep

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Mini Prep

Procedure for use of Zymo Research ZR Plasmid miniprep -Classic

Buffer Preparation:

  • This should be done the first time the kit is opened
Add 95% ethanol to the Plasmid Wash Buffer at a 4:1 Volume
Product number:
D4015=96mL of ethanol to Plasmid Wash Buffer
D4016=192mL of ethanol to Plasmid Wash Buffer
D4054=192mL of ethanol to each 48mL Plasmid Wash Buffer

Protocol

1) Centrifuge 0.5-5mL of bacterial culture in a clear 1.5mL tube at 15,000rpm for 20 seconds.

2) Add 200μL of P1 Buffer (red) to the 1.5 mL tube and resuspend pellet completely (vortex)

3) Add 200μL 0f P2 Buffer (green) and mix by inverting the tube 5 times. Cells are completly lysed when solution appears clear, purple, and viscous. Proceed to the next step withing 1-2 minutes.

4) Add 400μL of P3 Buffer (yellow) and mix gently but thoroughly. DO NOT VORTEX!!! The sample will turn yellow when the neutralization is complete. Allow the lysate to incubate at room temperature for 1-2 minutes.

  • P3 Buffer should be stored at 4°C.

5) Centrifuge samples for 2 minutes.

6) Place a Zymo-Spin IIN column in a Collection Tube and transfer the supernatant from step 5 into the Zymo-Spin IIN column.

  • When pipeting the supernatant, be careful not to disturb the green pellet to avoid transferring any cellular debris to the column.

7) Centrifuge the Zymo-Spin INN/Collection Tube assembly for 30 seconds.

8) Discard the flow through in the Collection Tube, making sure the flow through does not touch the bottom of the column. REturn the Zymo-Spin INN column to the Collection Tube.

9) Add 200μL of Endo-Wash Buffer to the column and centrifuge for 30 seconds.

10) Add 400μL of Plasmid Wash Buffer to the column. Centrifuge for 1 minute.

11) Transfer the column into a clean 1.5mL microcentrifuge tube and then add 30μL of sterile deionized water. Centrifuge for 30 seconds to elute the plasmid DNA.

12) Measure and record the concentration of the recovered plasmid on the nano-drop. (ask someone for help if it is your first time operating the nanodrop)

13) Dilute the plasmid to 50ng/μL with sterile deionized water and store in -20°C freezer.



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