Note: We found it is often necessary to add extra fresh RNase to the P1 buffer to prevent excessive RNA contamination using the simplified version of miraprep. Thus, we have stopped using miraprep as our routine DNA prep.

Miraprep uses a simple trick to greatly improve the DNA yield using a miniprep kit. From 2 mL terrific broth (TB) culture, we routinely get 30-50 µg plasmid DNA. For comparison, the average yield of a regular miniprep from 2 mL TB culture is 10-20 µg.

If you are very familiar with miniprep, the miraprep trick is to add an equal volume of ethanol to the supernatant before loading it onto the column, whereas all other steps remain essentially the same. The ethanol is thought to overcome the typical binding capacity of silica columns by condensing DNA into micro-precipitates.

1. Reagents

We usually use the Qiagen miniprep kit (Qiagen, 27104) that contains all required solutions and columns. However, you can also make all solutions according to the OpenWetWare recipe and use cheaper columns.

2. Procedure

1. Inoculate each bacterial colony into 2-4 mL terrific broth with appropriate antibiotics in a 14 mL round bottom Falcon tube. Culture in a 37°C shaker incubator at 250 rpm overnight.

2. Pour bacterial suspension into pre-labeled 2 mL tubes (eppendorf, catalog # 022363352). Centrifuge at 9,000 rpm for 1 min to pellet bacterial cells. Discard the supernatant by pouring into a liquid waste container.

   • If more than 2 mL was used, split into multiple 2 mL tubes.
   • For intermediate tubes, simply label them with 1, 2, 3 … can save a lot of labeling time, especially when processing many samples. Just make sure plasmids are ordered in a natural way to avoid trouble of decoding at the last step.

3. Add 250 µL P1 resuspension buffer (with 100 µg/mL RNase A) into each tube.

   • Extra fresh RNase A may need to be supplemented to minimize RNA contamination in the final prep.
   • When processing multiple tubes, open all tubes on a rack and use a repeater pipette can be a time-saver.

4. Resuspend the pellet by vortexing or scratching each tube against a tube rack for a few times. Although scratching can be annoyingly noisy, it is much faster.

5. Add 250 µL P2 lysis buffer into each tube. Invert the tubes 4-6 times or more until the solution becomes viscous and somewhat clear (it will never become very clear if too much bacteria were used – avoid using too much bacteria for each prep).

   • If LyseBlue was included in P1, the solution should turn blue.
   • Again, use a repeater pipette if processing multiple tubes.
   • When the amount of bacteria is on the higher end, you often need to wait for a couple minutes for the lysis to complete. However, do not go over 5 min for lysis as it may affect the recovery of plasmids.

6. Add 350 µL N3 neutralization buffer into each tube. Immediately invert the tube 4-6 times (or more) to make sure the solution was thoroughly mixed.

   • If LyseBlue was included in P1, the blue color should disappear.
   • I usually do not use the repeater pipette at this step to avoid localized precipitation in the solution (not sure how much it matters though).

7. Centrifuge at ≥ 13,000 rpm for 10 min to pellet cell debris.

8. During centrifugation, prepare a new set of 2 mL tubes on a rack and label them.

9. After centrifugation, pipet 800 µL supernatant into the new tubes. Try to avoid precipitates as much as possible.

10. Centrifuge again at ≥ 13,000 rpm for 5-10 min to pellet carryover precipitates.

    • During the centrifugation, prepare a new set of 2 mL tubes and a set of spin columns following the next 2 steps.
    • Carryover precipitates appear to be the main source of contamination that will give inflated measurements of DNA concentration.

11. Arrange a new set of 2 mL tubes on a rack, add 800 µL ethanol into each tube, close the tubes and label them.

12. Label a set of spin columns on the columns themselves (instead of on the waste collection tubes).

    • If processing multiple tubes, use a vacuum manifold can be a time-saver. Place labeled columns on the vacuum manifold.

13. After spinning, pour the supernatant into label-matched tubes containing 800 µL ethanol. Invert 4-6 times to mix.

14. Load half of the supernatant/ethanol mix onto a spin column by pouring. Centrifuge at ≥ 13,000 rpm for 1 min or use vacuum to drive it through the column. Repeat for the remaining half of the mix.

15. Wash each column using 750 µL PE buffer.

    • Since the volume here does not need to be precise, I often use a 10 mL or 25 mL pipette to fill up the columns with PE buffer when using a vacuum manifold.

16. Centrifuge emptied columns and collection tubes at ≥ 13,000 rpm for 1.5 min to get rid of residual PE buffer.

17. During the centrifugation, arrange a new set of 1.5 mL tubes on a tube rack. Do not close the tubes or label them yet.

18. After the centrifugation, place the columns onto the new set of 1.5 mL tubes. Add 50-75 µL EB buffer to each column. Let sit for ≥ 1 min.

19. Place the tubes in the centrifuge with the open lids towards the center. Put on the centrifuge lid and tighten it. Centrifuge at 9,000 rpm for 1 min in to elute the DNA.

20. Remove the columns and label the tubes one by one. This is when you can elaborate the labeling to your like.

21. Measure DNA concentrations on a nanodrop spectrometer. Perform diagnostic digestion test and send for Sanger sequencing if this is a newly constructed plasmid.