Miraprep: >30μg of Plasmid DNA Using a Miniprep Kit Every Time

First of all, @Michael Paulsmeyer had a great post a while back on how to get as much DNA from a standard miniprep as possible (https://www.scifind.net/methods/efficient-miniprep-spin-column-k). The variance in my personal results from minipreps is large, though. To make sure I always get a high concentration of DNA, I use a ‘Miraprep’ protocol adapted from this paper: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160509. This essentially allows you to get maxiprep-level concentrations from a miniprep kit. Using this ensures that you will get >30μg of plasmid DNA, and the protocol is only about 20 minutes longer than a standard miniprep.

You can use a lot of different kinds of kits for this (as discussed in the paper), but here is what I do step-by-step with a GeneJET Plasmid Miniprep Kit from Thermo Fisher:

Day 1 (Bacterial Overnights)

-Suspend colonies of plasmid in 50mL of LB (and drugs for selection if necessary) and leave to grow overnight at 37C w/ shaking. I try to add a lot of colonies from the corresponding plate so that it will grow up a lot by the next day (so this might not be as effective straight from a transformation when you only have one colony, but it might still work).

Day 2 (Miraprep)

  1. Centrifuge the 50mL overnight to pellet.
  2. Discard the supernatant and add 2mL resuspension buffer from the miniprep kit- pipette up and down to resuspend.
  3. Add 2mL of lysis buffer from the miniprep kit and invert ~10 times to mix. Incubate at room temperature for 2 minutes, and no longer than 5 minutes.
  4. Add 2mL of neutralization buffer from the miniprep kit and invert ~10 times to mix.
  5. Pour the contents of the 50mL tube into four different 1.5mL micro-centrifuge tubes.
  6. Spin the four tubes at 13 000 x G for 10 minutes to pellet.
  7. Take the supernatant from all four tubes and add them into one 15mL falcon tube.
  8. Add ~5mL of 95% ethanol to the 15mL tube (ie. 1X the amount of supernatant already in the falcon tube), and vortex to mix thoroughly.
  9. Load 700μL (no more) of the mixture into one GeneJet column from the miniprep kit.
  10. Spin the column at 13 000 x G for ~30s.
  11. Repeat steps 9 and 10 until no mixture remains (will need to be done ~10-15 times).
  12. Add 500μL of wash buffer to the column.
  13. Spin the column at 13 000 x G for ~30s.
  14. Repeat steps 12 and 13 once.
  15. Centrifuge the empty column for 1.5 minutes to dry. Place the columns in new microcentrifuge tubes.
  16. Add 40-50μL elution buffer OR water to the column (warm the elution buffer/water to 60°C prior to adding it).
  17. Incubate the column at room temperature for 2 minutes.
  18. Spin the column at 13 000 x G for 2 minutes to elute the DNA.

Expect concentrations from 700-1000ng/μL. If >40μg of DNA is required, consider making two 50mL overnights and running the protocol twice (simultaneously).

The other cool thing about this is that sometimes instead of doing a 50mL overnight I will just do two or three 5mL overnights, follow the standard miniprep protocol with all of them individually, BUT once I get to the step where you transfer the supernatant to a column following lysis and neutralization, I transfer one supernatant at a time in-between centrifuges to the same column. It’s the same idea as what you’re doing in a Miraprep except it saves on reagents and it's quicker- it only adds an extra centrifuge or two to the standard miniprep protocol.

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this sounds amazing - I'll definitely give it a go when I need lots of plasmid DNA next time! I'm guessing the usual size restrictions on plasmid DNA still apply though, so I'm best off using midi or maxipreps for large plasmids?

Nick Gervais2 months ago

@David Walker-Sünderhauf yeahhhhh I imagine since you're just using a normal miniprep kit you'd still have to follow the rules for whatever kit you're using

Michael Paulsmeyer2 months ago

Wow that is great yield! I can't believe those mini GeneJet columns can hold so much DNA.

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