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How to: Get Better Plasmid midiprep Yields

How to: Get Better Plasmid midiprep Yields

I get many people complaining to me about poor DNA yields from commercial plasmid plasmid prep kits. In this article I will explain the main pitfalls in plasmid isolation and how to avoid them.

Get the culture prep right

You will always get the best results by taking care in preparing the culture. Innoculating from old colonies or allowing the culture to become too saturated will result in poor plasmid replication and retention, which could severely affect the yield. In the past grew my midiprep cultures overnight so they were completely saturated in the morning, but since I switched to the following procedure, my yields have improved dramatically:

  • Start with a fresh colony, no more than a few days old.
  • Innoculate a 5 mL LB starter culture with the colony in the morning and grow until the OD600 is approximately 1, then store in the fridge overnight.
  • If you are using ampicillin as the selection vector, pellet the cells and resuspend in fresh, antibiotic-free medium to remove the secreted beta-lactamase.
  • Next morning innoculate a 100 mL LB culture with 1 mL from the starter culture for high copy number plasmids, double the volumes for low copy plasmids.
  • Grow the culture until OD600 is around 3, then pellet the cells and proceed directly to the miniprep, or freeze the cells at -2o°C and continue at a later time.

Make the lysis and neutralization thorough

The biggest mistake people make in midipreps is with the mixing in the lysis and neutralization steps. The protocols normally emphasize the importance of being gentle to prevent shearing of the genomic DNA, but I find that people tend to be too gentle. Another common problem is that the lysis allowed to proceed for too long, resulting in permanently denatured, undigestable DNA. Here’s how I approach lysis and neutralization:

  • If you are using a low copy number plasmid, use double the recommended volumes of re-suspension, lysis and neutralization buffers. This might also help for high copy plasmids that have given low yields or poor lysis in the past.
  • After adding the lysis buffer, instead the 4-6 inversions recommended in the protocols, gently mix by inverting the tube continuously for 3 minutes then immediately add the neutralization buffer.
  • Mix the neutralization buffer by gently inverting continuously for 1 minute.
  • If the precipitate looks like desiccated coconut, then things are looking good. If it looks gloopy, this is bad. Try to break up the gloop by mixing, this time a little more vigorously (but still quite gently) for a further 1 minute.
  • If the glop still does not break up, then the yield is likely to be poor. Next time use double buffer volumes as described above.

Protect the column, and elute twice

Just a couple more quick tips.

  • If you are using centrifugation to remove the precipitated debris, filter the supernatant though Whatmann paper before applying it to the column since small amounts of precipitate could easily block the column.
  • Perform the elution twice (into separate collection tubes) since a lot of DNA can be left on the column after the first elution. Using elution buffer heated to 50°C can also help to increase elution yield.


  1. Isobel on July 10, 2018 at 7:20 pm

    I am trying to isolate plasmid from agrobacterium. There are two different plasmids both are within the same agrobacterium strain. When I isolate them side by side I get good results from one and no plasmid yield from the other although everything looks good during the process. Any guess and what could be the problem

  2. mariana on January 26, 2018 at 7:33 pm

    Hi, can anyone tell me which type of watman paper should I use for filtration of bacteria lysis product before aplying to the column

    • Mark Bergseid on November 11, 2019 at 2:58 am

      almost anything, 3M is common and good,

  3. Steve G on March 24, 2017 at 7:13 pm

    Thanks for the tips! Do all apply to miniprep protocols as well?

  4. MAPAYA GIVEN on July 1, 2016 at 8:29 am

    please anyone help me
    i want to isolate plasmid dna from my e.coli cells so i want to know how the OD affect the amount of plasmid that i will isolate,if i have high OD what will be the effect compare to that one with low OD?

    • Dr Amanda Welch on July 1, 2016 at 5:23 pm

      Either a high or low OD600 can adversely affect your isolation. If you have too high of an OD600, then you’ll overwhelm the chemistry of the kit (and you’ll get a lot of junk in your eluent). If your OD600 is too low, then there won’t be a lot of bacteria to get your plasmid from.

  5. joshua ifeoluwa on April 21, 2016 at 7:51 pm

    I think I will try out this technique not saying my usual method of inoculating overnight culture doesn’t work but I will compare

  6. Tierney on April 11, 2016 at 11:57 am


    I’ve been attempting to extract plasmids from my E. coli clones recently but have had very poor recovery (10-20 ng/uL) and would like to try this method but I have a few questions.

    Why is it recommended to refrigerate the 5 mL LB culture grown to OD600 ~1 overnight before inoculating the next culture? And when growing the second culture, is an OD of 3 very high, I thought 0.4 – 0.6 would be representative of the log phase of growth?

    Any help would be much appreciated, thanks!


    • ZD on March 3, 2017 at 10:11 am

      Hi, lately I have been facing this same problem.. my plasmid yield is too low (20-60ng /microlit). Were you able to solve your problem ?
      How did you manage.. ?

  7. tara on February 3, 2016 at 7:03 pm

    For large DNA constructs such as BACs, I use Nucleobond columns from Clontech.

  8. Rivu on December 3, 2015 at 5:42 pm

    Hey, thanks for the tips 🙂
    I wanted to know if you have any experience with the use of frozen pellets in midi/maxi prep. By frozen pellets i mean spinning down bacteria and getting rid of the medium after incubation. Then snap freezing in -80 freezer. using this pellet to resuspend the bacteria for extraction, would it be a good idea? And how do you think it would affect the yield in comparison to freshly pelleted bacteria?

    • Dr Amanda Welch on December 4, 2015 at 12:43 am

      I’ve done this with bacterial cells that were at -20C for weeks. I didn’t see an appreciable difference in yield. That being said, my plasmids were generally around the 3.5-4 kb range.

  9. michael rak on May 13, 2015 at 8:35 pm

    Err… pH 7.0, if need be.

  10. michael rak on May 13, 2015 at 8:30 pm

    Can anybody explain why midiprep (DEAE functionalized resin columns) elution buffers contain isopropanol (qiagen, machrey-nagel xtra) or ethanol (machrey-nagel AX-100 and classic midiprep series) in the elution buffer? I’ve been looking for a suitable answer, and can’t find one.

    Also, any tips for eluting >200kb product off midiprep columns? I’ve tried the usual tips from the manuals: warming the elution buffer, incubating and eluting at 65C or greater, etc. I’m considering trying to elute with 1.8M KCl pH 8.0, but I don’t know the function of an alcohol (isopropanol or ethanol) in midiprep elution buffers.

    I’ll paypal a dollar to the first person with an impressive and satisfying answer.

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