Plasmid miniprep

Key Concepts

A miniprep is a technique that allows us to isolate plasmid DNA from bacterial cultures. The isolated DNA is high-purity and can be used for sequencing, digestion, or PCR. Like for PCR cleanup and gel extraction, most labs use kits to do minipreps. 

Minipreps are often ideal for basic cloning, since they only require small volumes of cultured cells and produce plenty of high-quality DNA for most downstream applications. There are also Midiprep and Maxiprep kits that produce greater volumes of purified DNA, but they require much larger bacterial cultures. During  the miniprep, we will lyse the bacteria grown in culture from the previous day and purify the transformed plasmids from them. 

The afternoon before the miniprep, use a pipette tip to inoculate 3 – 5 mL of LB media + 1x kanamycin with a single colony from the transformation of your Gibson reaction. Incubate the culture overnight at 37 °C in a shaking incubator (typically ~250 rpm).

Materials

• 1.5ml microcentrifuge tubes
• Plasmid Miniprep kit

Procedure

Centrifugation should be performed at approx. 10,000g unless otherwise specified.

Note: Instructions provided are for the Zymo miniprep kit. While all miniprep kits utilize similar protocols, there may be small but significant differences between manufacturers. If using another kit, please follow the instructions listed by the manufacturer for steps 2-11.

1. Centrifuge 1-5mL of bacterial culture at maximum speed in a 1.5mL microcentrifuge tube. Because the 1.5mL tubes can only hold ~1.4mL at a time, repeat as necessary to pellet all of the culture. Discard the supernatant after each spin.
2. Add 600uL water or Zymo Resuspension buffer to the bacterial cell pellet and resuspend completely.
3. Add 100ul of 7X Zymo Lysis buffer (blue) and mix by inverting the tube 4-6 times. Proceed to step 4 within 2 minutes. After addition of the lysis buffer, the solution should change from opaque to clear blue, indicating complete lysis.
4. Add 350uL of cold Zymo Neutralization buffer (yellow) and mix thoroughly by inverting the tube 4-6 times. The sample will turn yellow when the neutralization is complete. Invert sample an additional 2-3 times to ensure complete neutralization.
5. Centrifuge at maximum speed for 2-4 minutes.
6. Transfer the supernatant (~900uL) to a provided Zymo-Spin IIN column in a collection tube. Avoid disturbing the cell debris pellet. This can be done by pipetting or careful pouring.
7. Centrifuge 15 seconds and discard the flow-through. Place the column back in the collection tube.
8. Add 200uL of Zymo Endo-Wash buffer to the column. Centrifuge for 30 seconds. It is not necessary to empty the collection tube after this spin.
9. Add 400uL Zyppy Wash buffer to the column. Centrifuge 2 minutes.
10. Transfer the column into a clean 1.5mL tube labeled “Plasmid miniprep.” Add 30uL water directly to the column matrix and incubate 1 minute at room temperature.
11. Centrifuge 30 seconds to elute plasmid DNA.
12. Quantify DNA concentration by Nanodrop. Record the concentration on the side of the tube.

Store DNA at 4C until ready to use.