The Miraprep: A Protocol that Uses a Miniprep Kit and Provides Maxiprep Yields
Authors: Mark Peifer
Natalie Deuitch
Mira I Pronobis
Journal: PLoS ONE (2016)
Discipline: Multidisciplinary
Keywords: Plasmid purification; Miraprep; DNA isolation; Miniprep kits; Maxiprep
Affiliations: Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America., Department of Biology, University of North Carolina at Chapel Hill, CB#3280, Chapel Hill, NC, United States of America.

Abstract:  Plasmid purification is a basic tool of molecular biologists. Although the development of plasmid isolation kits utilizing silica spin columns reduced the time and labor spent on plasmid purification, achieving large plasmid DNA yields still requires significant time and effort. Here we introduce the Miraprep, a rapid protocol that allows isolation of plasmid DNA using commercial Miniprep kits, but with DNA yields comparable to commercial Maxiprep plasmid purifications. Combining ethanol precipitation with spin column purification, we created a DNA isolation protocol that yields highly concentrated plasmid DNA samples in less than 30 minutes. We show that Miraprep isolated plasmids are as stable as plasmids isolated by standard procedures, can be used for standard molecular biology procedures including DNA sequencing, and can be efficiently transfected into mammalian cells. This new plasmid DNA isolation protocol will significantly reduce time and labor without increasing costs. 

Retracted: No

PubPeer comments:
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 I appreciate the authors effort in promoting efficiency in the scientific community; however, I concur with Peer 1 s assessment. I performed the Miraprep protocol as written (using Sigma GenElute columns) and, in parallel, omitted the ethanol step. My nanodrop quantification suggested 10x increased yield with the ethanol addition, yet both samples look roughly the same on a gel. The only discernible difference is that the preps including ethanol have a diffuse band running lower than my 500 bp marker. I find the most likely explanation is that the ethanol causes a UV-absorbing contaminant, possibly a low molecular weight nucleic acid, to be retained on the column. In line with the authors mission of promoting efficiency in the scientific community, I encourage them to repeat my experiment and address this disparity in their study. A retraction or published erratum may save others from overestimating their DNA yields or confounding downstream applications by this method. , 
 1. (-) ethanol. Nanodrop estimate 262.8 ng uL. 2. (+) ethanol. Note low molecular weight smear. Nanodrop estimate 4,716.3 ng uL. ![file] , 
 #3 Probably, the high readings are from RNA. I had a similar effect in an unrelated protocol (selective DNA precipitation) when decided to omit RNAse at the first step: got really high yields according to NanoDrop due to the residual RNA. Though, the effect was particularly pronounced for low-copy plasmids. , 
 #4 Yes, good point. The ethanol probably precipitates partially digested, low molecular weight RNA (which is normally too small to stick to the column and remains soluble), causing it to bind to the column and skew the subsequent nanodrop reads. , 
 Frequently encountered problems with the Miraprep: Presence of RNA and or low yield: The issue of potential RNA contamination was raised by the Reviewers during the Review process. The reviewers requested and we carried out a number of additional control experiments to address this issue. In the published version of our manuscript we extensively investigated the presence of RNA in the sample by measuring the OD 260 280 ratio, adding different concentration of RNase A to the samples, running gels to test for low molecular weight RNA, and comparing standard and Miraprep prepped plasmids on agarose gels. Since this was a potential issue, we made certain to emphasize that RNA contamination can be avoided by adding RNase A fresh every time at a concentration of 50ug ml in the resuspension buffer. This is critical for the successful DNA yield described in our paper. We understand that it is tempting to just take the old miniprep kit and follow the Miraprep protocol without addition of RNase A, but this can generate plasmid isolations with RNA contamination. In fact RNase A activity in the resuspension buffer decreases dramatically after 2 weeks (a fact also noted by most manufacturers). We found that 50ug ml of RNase A added fresh at the beginning of the protocol was sufficient to give us high DNA yields with a normal 260 280 ratio as described in the paper. We can not exclude that columns from other manufacturers wonu2019t yield the same results as the 2 miniprep kits from GeneJet and Qiagen. We also recommend to use the suggested amount of volume of bacteria culture per column (10ml bacteria culture column). Further as mentioned in our manuscript, contamination of the sample by things other than nucleic acids or proteins might alter the OD 260 280 ratio. However, we documented in our paper that for the purpose of sequencing, restriction enzyme digests, and mammalian cell culture transformation, Miraprep isolated plasmids worked well. A colleague from another lab recently informed us that he injected zebrafish embryos with Miraprep isolated plasmids, and he stated that he had a better yield of positive embryos than with his phenol-chloroform isolated plasmids. We had not tested this possibility when we wrote our manuscript, but it appears that the Miraprep DNA are clean enough to allow injected zebrafish embryos survive. In conclusion to avoid the common problems, we strongly recommend that experimenters add 50ug ml RNase A fresh each time into the resuspension buffer, keep the suggested bacteria culture volume of 10ml column, and use the miniprep kits that have been tested in the manuscript. , 
 The Miraprep I am curious about the naming: is it after the first author, someone else with this name, the MIRA grant or some kind of abbreviation? P.S.: Oh, I ve found it: Comparison of commercial plasmid preparation methods from three manufactureru2019s (GeneJet, Qiagen, and GenElute) with the **Miracle-prep**. It s a miracle-prep. , 
 Here is the data associated with my comment (see Maria E Gallegos) ![file] , 
 #6 I was very excited about a potential improvement to the miniprep particularly since I need large amounts of a plasmid that grows in ccdB survival cells. I tried the miracle prep several times (1st time: freshly added RNAse A that came with the Qiagen prep. 2nd time: Freshly added RNAse A (ThermoFisher EN053) to a final concentration of 50 ug ml. 3rd time: Freshly added RNAse A (ThermoFisher EN053) to a final concentration of 100 ug ml. As expected from the publication, I observed a nearly 10X increase in yield (by nanodrop) but the A260 A280 ratio never went below 2.0. For the final series of preps (normal and miracle done side by side), I digested 150 ng of the normal prep (-1) and  150 ng of the miracle prep (-2) then analyzed by gel electrophoresis. The miracle prep came out to be about 8X less intense (quantified by FIJI). Not sure what is going on but this prep is not working for me. , 
 1) Could the authors please state more explicitly what *E. coli* strain was used for the pDNA preparations. I wasn t able to find this in the paper (but maybe it is in there somewhere and I didn t look hard enough). The yield from different strains varies quite a lot. 2) It would be useful to state not just the culture volume but also the wet cell weight of the pellet (commonly stated for protein purifications). This way it is more straightforward and easier to compare different studies and protocols (by DNA yield per mg wet weight). *E. coli* cell density in the media can vary considerably depending on a vessel, culture volume per vessel, shaking method, E. coli strain, plasmid, (sometimes) trypton supplier, etc. Therefore yields per ml culture are not exactly comparable. 3) Interestingly, the QIAquicku00ae Gel Extraction Kit From Qiagen recommends adding 1 gel volume of isopropanol (usually =20% volume) to improve the DNA yield for fragments 500 bp and 4 kb. I wonder why they didn t expand this approach then to the pDNA kits. 4) The choice of DNA markers is very strange on many figures. The plasmids are 3kb, 8kb and 14 kb while the marker often ends way below the pDNA bands. 5) Fig. 2. On these DNA gels, the amount of purified DNA is larger than the initial amount in the lysate. Naturally, I would expect that the volumes put on the gel are chosen to reflect % yield at each step (thus, smaller DNA amount for the final preparation). Apparently, the DNA amounts to apply were chosen differently here. ![file] , 
 #6 In conclusion to avoid the common problems, we strongly recommend that experimenters add 50ug ml RNase A fresh each time into the resuspension buffer RNAse A is one of the most stable enzymes in existence and the DNAse-free version is commonly obtained by boiling. Unless there is some microbial contamination, the RNAse-containing solutions retain their functionality for months at least and addition of fresh RNAse each time seems not to be warranted. , 
 I m not able to get rid of the RNA during lysis I have taken the author s advise and added fresh RNAse (despite rnase working at 10ug ml in my non mirapreps). I am using LB24 which is a very rich broth and makes heaps of plasmid it seems like using rich broths is a known issue from my Twitter searching https: twitter.com fe_emiliani status 1523639734399995906. I m also doing a heat shock for 2hrs in line with the original mutant ColE1 paper I think most people have forgotten that high copy number mutants of ColE1 and the pUC ori can make heaps more with this one trick. To demonstrate to the authors that this is not a simple case of adding more RNAse I added 300ug ml of brand new 3000u mg RNAse, to no avail, attached is the same plasmid mirapreped with 50ug of RNase (left) and 300ug ml of RNase (right). SDS is an inhibitor of RNAse, perhaps an on column RNA digest in a high chloride buffer (1M NaCl 50ug ml RNAse) would work but you could also just not do a miraprep and save yourself some time and still be able to use lower volumes or richer media for bulk plasmid! ![file] ,