1.2: Plasmid DNA Extraction (Mini-Prep)

Zymo-Pure Plasmid DNA extraction (Micro-Centrifuge Method)

 

Learning Objective

• Isolation of Plasmid DNA from overnight cultures in LB.

This method is spin column-based and purifies up to 100 $\mu g$ of ultra-pure endotoxin-free plasmid DNA in less than 15 minutes. The result is plasmid DNA suitable for transfection, restriction endonuclease digestion, bacterial transformation, PCR amplification, and DNA sequencing. (ZymoPURE Plasmid Miniprep Kit)

Note

Watch the Youtube video for questions about the Miniprep protocol

ZymoPURE Plasmid Miniprep video

Note

The P1 reagent is temperature sensitive due to RNase being present, and should be kept in the fridge or on ice at all times. All other reagents will be stored at room temperature.

Reagent name and Code	Storage Temperature
ZymoPURE™ P1 (Red)	4°C
ZymoPURE™ P2 (Green)	Room Temp.
ZymoPURE™ P3 (Yellow)	Room Temp.
ZymoPURE™ Binding Buffer	Room Temp.
ZymoPURE™ Wash 1	Room Temp.
ZymoPURE™ Wash 2	Room Temp.
ZymoPURE™ Elution Buffer	Room Temp.
Zymo-Spin™ II-P Columns (must have purple ring)	Room Temp.
Collection Tubes	Room Temp.

Visual Procedure of Zymo Plasmid Mini-Prep

TIPS BEFORE STARTING

The first couple of times you do the Mini-Prep, on the protocol indicate/LABEL WHERE your DNA is for each of the steps! (e.g., DNA is in the supernatant/liquid OR DNA is in the pellet). Each step below has a “circle one” option asking where the DNA is! (pellet or supernatant).

Note

If you understand exactly where your DNA is in each one of the steps, you won’t LOSE your DNA!!

Protocol: (make sure you have all reagents before you begin… get ice!!!)

1. Using a serological pipet, pipet out 1.0 to 3 mL (preferably 3 ml) of bacterial culture into a 1.5 ml microcentrifuge tube. Centrifuge this microcentrifuge tube at full speed for 20 seconds. You will see a small pellet form at the bottom of the tube. (You must repeat steps 1-2 to get more than 1 ml worth of cells
   
       Where is your DNA?           Pellet or          Supernatant
    
2. Remove as much media as possible by pouring it off into the Biohazard bag. Do not disrupt the cell pellet! (Repeat steps 1-2 to increase cell concentration: However, 1-2ml of culture should be enough plasmid.)

Where is your DNA?           Pellet or          Supernatant

3. Add 250 $\mu$L of ZymoPURE™ P1 (Red) to the bacterial cell pellet and vortex or pipet the pellet to resuspend it completely. (P1 is stored in the fridge at 4°C. Keep on ice when not in use.) This step involves RNase to purify the DNA by destroying any RNA in the sample.

Where is your DNA?           Pellet or          Supernatant

4. Add 250 $\mu$L of ZymoPURE™ P2 (Green) and immediately mix by gently inverting the tube 6-8 times. Do not vortex! Let sit at room temperature for 2-3 minutes. Do not let sit for longer than 3 minutes. (Cells are completely lysed when the solution appears clear, purple, and viscous.)

Where is your DNA?           Pellet or          Supernatant

5. Add 250 $\mu$L of ice cold ZymoPURE™ P3 (Yellow) and mix thoroughly by inversion. Do not vortex! Invert the tube an additional 3-4 times after the sample turns completely yellow. (The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form.)
   
   Where is your DNA?           Pellet or          Supernatant

6. Incubate the neutralized lysate in the microcentrifuge tube on ice for 5 minutes
   
   Where is your DNA?           Pellet or          Supernatant

• Centrifuge the neutralized lysate for 5 minutes at 16,000 xg (or Max speed). (Do not disrupt the white/pelleted cell debris).

  Where is your DNA?           Pellet or          Supernatant

8. Transfer 600 $\mu$L of supernatant from step 7 into a clean 1.5 ml microcentrifuge tube. Be careful not to disturb the yellow pellet and avoid transferring any white cellular debris to the new tube.
   
   Where is your DNA?           Pellet or          Supernatant

9. Add 275 $\mu$L of ZymoPURE™ Binding Buffer to the cleared lysate from step 8 and mix thoroughly by inverting the capped tube 8 times.
10. Place a Zymo-Spin™ II-P Column (Purple ring) in a Collection Tube and transfer the entire mixture from step 9 by pouring or pipetting it into the Zymo-Spin^TM II-P Column.
11. Incubate the Zymo-Spin™ II-P/Collection Tube assembly at room temperature for 2 minutes and then centrifuge at 5,000 xg for 1 min. Discard the flow through in the Biohazard bag.

12. Add 800 $\mu$L of ZymoPURE™ Wash 1 to the Zymo-Spin™ II-P Column and centrifuge at 5,000 xg for 1 min. Discard the flow through.
    (The collection tube will hold 900ul of liquid. It is important that the flowthrough does not touch the bottom of the column!)

13. Add 800 $\mu$L of ZymoPURE™ Wash 2 to the Zymo-Spin™ II-P Column and centrifuge at 5,000 xg for 1 min. Discard the flow through.
14. Add 200 $\mu$L of ZymoPURE™ Wash 2 to the Zymo-Spin™ II-P Column and centrifuge at 5,000 xg for 1 min. Discard the flow through.
15. Centrifuge the Zymo-Spin™ II-P Column at $\ge$ 10,000 xg (full speed) for 1 min in order to remove any residual wash buffer.
16. Transfer the Zymo-Spin™ II-P Column into a clean 1.5 ml microcentrifuge tube and add 25 $\mu$L of (warmed 50$^{\circ}C$- optional) ZymoPURE™ Elution Buffer directly to the column matrix. Incubate at room temperature for 2 minutes, and then centrifuge at $\ge$ 10,000 xg (full speed) for 1 minute in a microcentrifuge. (warmed buffer and a 5 min incubation may increase plasmid concentrations)

17. Determine the concentration of your sample using a spectrophotometer (E.g. Denovix, NanoDrop, Qubit). (See appendices II, III, IV on how to use one of the aforementioned machines.)
    
    DNA Purity: Eluted DNA is ultrapure, endotoxin-free, and well suited for transfection, transformation, sequencing, restriction endonuclease digestion, in vitro transcription, and other sensitive applications.
    Typical: Abs 260/280 $\ge$ 1.8 and Abs 260/230 $\ge$ 2.0

18. Clearly label each plasmid tube; store it at -20 degrees and record it in the lab notebook!
    
    What should be on your label?

Top label:

Part # (Bba_J0176)

Side label:

Team Name/Initials (SV/JH Team)

Part Name (RBS: GFP)
Concentration (ng/µL) 260/280 ratio

Plasmid Backbone (Antibiotic Resistance) / psB1C3 (Chlor)
(Date) 6/14/2021