Plasmid v Genomic DNA Extraction:The Difference

Avatar of Suzanne Kennedy
About Suzanne Kennedy
Suzanne is Director of R&D at Mo Bio Laboratories in California, and the author of their blog, The Culture Dish. She has a PhD in Microbiology and Immunology from Virginia Commonwealth University.

If you want to isolate plasmid DNA, you crack your cells open and carry out a miniprep, trying very hard not to get any contaminating genomic DNA in your sample. If you want genomic DNA, you crack your cells open in a different way and try to isolate as much of the stuff as possible.

So what's the difference?

In this article, I'll explain how both plasmid and genomic DNA preps work and how they are different.

Genomic DNA Extraction:

1. Lysis: Just Crack Them Open

Genomic DNA extraction is the simpler of the two procedures because all that is needed is a good strong lysis to release the genomic DNA into solution. For yeast, plant cells and bacteria, this involves breaking down the strong, rigid cell wall before mechanically disrupting the membrane. The cell wall can normally be broken down using enzymes such as lysozyme, which catalyses the hydrolysis of the cell wall peptidoglycans and the serine protease, proteinase K (and for gram+ species, lysostaphin will help). For more exotic species with different cell wall compositions, different enzymes may be required.

A more universal method of lysis for genomic DNA extraction involves mechanically breaking the cell wall. One method for this is bead beating, which can be easily performed on a vortex using 0.1 mm glass beads or 0.15 mm fine garnet beads. Special vortex adapters help with performing multiple extractions at the same time with equal efficiency. Bead beating is faster than enzymatic lysis and generally more thorough.

2. …and purify

Once the sample has been lysed so bringing the genomic DNA into solution, all that is needed is to purify the sample. This can be achieved using either phenol-chloroform or a spin filter membranes by adding guanidine salts that promote binding to silica.

3. Some words of advice

The chromosome is going to break during purification because it is much too big to stay in one piece. But for most applications this is not a problem and for PCR or qPCR, the breakage will be an advantage because it allows better melting the DNA and result in a more efficient reaction.

The E.coli chromosome is 4,638, 858 bp long and this comes to roughly .005 picograms per cell. In a typical overnight culture started from a single colony, the bacteria number around 1-2×109 bacteria/ml. That means that 1 ml of culture should yield about 5 µg of genomic DNA per 109 bacteria.

Plasmid DNA Extraction

Plasmid DNA extraction is a bit more complicated because it involves separating the plasmid from the genomic DNA. The separation of the two forms of DNA is based on size…

…and the trick is in the lysis method.

1. Alkaline Lysis

For plasmid DNA extraction, the lysis has to be a lot more subtle than simply chewing up the cell wall with enzyme or bashing it with glass beads. The (virtually) universal method for plasmid DNA extraction was invented by Birnboim and Doly in 1979 (and was explained by Bitesize Bio in 2008!)

The lysis buffer contains sodium hydroxide and SDS, the purpose of which is to completely denature of the plasmid and genomic DNA (i.e. separate the DNA into single strands). It is critical that this step is performed quickly because too long in the denaturing conditions of this solution may result in irreversibly denatured plasmid at the end.

Next the sample is neutralized in a potassium acetate solution to renature the plasmid.

And this is the key to the separation of the plasmid and genomic DNA.

Because plasmid is small, it can easily re-anneal. But the genomic DNA is too long to re-anneal properly and instead it becomes tangled so the complimentary strands stay separated.

When the sample is centrifuged, the genomic DNA is still bound to protein and gets pulled down while plasmid DNA is soluble and free. It is key at this step not to vortex or mix the sample vigorously because the genomic DNA is easy to break, and broken genomic DNA can be small enough to re-anneal and go into solution with the plasmid.

2. Purification

The plasmid DNA is recovered in the supernatant and can now be ethanol precipitated for a crude prep or cleaned up using phenol-chloroform or a spin filter based prep. If you are using a spin filter prep, the neutralization buffer will already contain guanidine salts so the lysate can be bound directly onto silica for further washing and elution. The pure DNA is fine for most everything from cloning to sequencing. If the plasmid is to be used for transfection, anion-exchange purification is a better choice to remove the endotoxin, although endotoxin removal is available using faster silica based purification also.

The method for purifying plasmids can also be used for mammalian plasmids transfected in eukaryotic cells or for any other small extra-chromosomal DNA. The difference for mammalian cells or chloroplast/mitochondrial DNA is that the copy numbers are much smaller compared to the high copies of plasmid that can be obtained. So expect a lower yield if you try the plasmid method on another type of DNA isolation or scale up your buffer accordingly if you decide to start with more sample.

3. …and some words of advice.

Plasmid DNA is typically 3-5 kb and then the size is increased based on the insert. The type of origin of replication will affect how high the copy number will be per cell. A typical high copy number plasmid such as pUC or pBluescript should yield between 4-5 µg of DNA per ml of LB culture.

To isolate high yields of plasmid DNA, the culture should be in late log phase or early stationary phase. Prepare cultures using fresh single colonies from plates and make sure the antibiotic is fresh and the correct strength to maintain the plasmid during growth. It is important not to overgrow the culture or it may result in genomic DNA contamination in the plasmid prep.

Hopefully that was clear and helpful for you, but if not, you know what to do….

21 thoughts on “Plasmid v Genomic DNA Extraction:The Difference”

  1. Kurt says:

    The problem comes when your dealing with big plasmids up to 100 kb, that need to be separated from the chromosomal DNA.

  2. Hi Kurt,
    That is a good point to mention. Plasmids (BAC clones) up to 300 kb can be separated from genomic DNA successfully as long as you are very gentle with the mixing so you do not break the clone. I have isolated PI clones that were 150-200 kb on anion-exchange resins and with cesium chloride with good results.
    Only anion-exchange kits (such as Qiagen Maxi kits) can be used for plasmids greater than 50 kb.
    The main issue I had with large constructs is the low copy number and the need to scale up the culture to get a decent yield. Large constructs exist as <10 copies per cell so expect to recover around 0.1-0.2 ug of DNA per ml of culture (so 50 ug / 500 ml).

  3. dsbreak says:

    I noticed that some protocols (Epicentre, MO BIO) include a heating step at 65 degrees during the disruption of yeast cell walls. Any insight/references into how this works?

    1. The heating step serves to help weaken the cell walls before beating using beads. Heating denatures proteins and disrupts bonding, making it easier to break the cells with shearing forces.

  4. Alumi says:

    I need to extract genomic DNA from E.coli, but mostly I get a lot of plasmid DNA, too. I followed the protocols from "Current protocols in molecular biology" (unit 2.4), but I couldn't separate plasmid DNA from genome well. Which step I should notice to prevent plasmid when I extract genomic DNA? Thank you^^

    1. Hi Alumin,
      Is this in the absence of antibiotic? You should leave antibiotic out so that the plasmid has not pressure to be maintained.
      When you isolate genomic DNA, you isolate all the DNA in the cell. The best way to get rid of the plasmid is to isolate pure colonies that do not have it. This can be done by streaking colonies on antibiotic-less plates or growing in medium without it.
      If the E.coli strain doesn't matter and you just want cells without plasmid, you could try buying some competent cells (like XLIblue, DH5Alpha) and then plate them. They will have no plasmid.

      Best,
      Suzanne

  5. Avatar of Brian Cady says:

    "It is important not to overgrow the culture or it may result in genomic DNA contamination in the plasmid prep."

    Why is this? How does the stage of growth affect isolation of plasmid from gneomic DNA?

    Brian

  6. Hi Brian,
    When the culture reaches death phase, the cells will start lysing. This will lead to genomic DNA in the supernatant. If you can, image broken cell walls with DNA attached but hanging free. They are brought down with the bacterial pellet.
    However it is going to shear because it is long and also because of DNases, so you will have pieces of genomic DNA now mixed with the plasmid.
    Normally, the genomic DNA is discarded with the cell walls and protein after you precipitate everything in the third solution- but this requires that the DNA is intact. If you have broken bits of released genomic DNA floating around, it will be in your plasmid supernatant.

    So for plasmid preps, the best yield comes from end of log/early stationary. If you overgrow- either because you inoculated too heavy, grew too long, or used a rich media, you'll push the culture into death phase too soon and loose your plasmid and have a lot more genomic DNA.

    Best,
    Suzanne

    1. Hi Suzanne,
      does the fact that death phase is not good for plasmid extraction because of shearing of genomic DNA is true also for a cell culture pelletted and conserved at -20°C for some days?

  7. Avatar of Chris G Chris G says:

    Hi Suzanne,

    I need to extract large plasmids (and separate them from genomic DNA for sequecing) and I am considering using a CsCl method. Do you have a method that you would recommend?

    Chris

    1. Hi Chris,
      I have used the old fashioned cesium chloride preps before but I don't think I would recommend this. How big is your plasmid? I would try the Qiagen Large Construct Kit if they still sell it. It will be easier and it works well. I used it for 100 kb PI clones and it was much better than pulling plasmids out of cesium gradients. The yields will be low- probably around 0.1-0.2 ug/ml of culture.
      Since the recipes for the anion-exchange kit are published, you could make your own P1, P2, and P3 and then process large volumes of culture, precipitate down the lysate, and then add to a Midi column to maximize the yield.

      Best,
      Suzanne

  8. Avatar of m v m v says:

    what exp. do you guys have correlating biomass to plasmid yield? My last results were off the charts…

  9. Avatar of pijaw says:

    May i ask something?i recently conducted extraction of plasmid DNA from E.coli and one of the steps was to not vortex to avoid shearing genomic DNA. my question is-how does sheared genomic DNA contaminate the sample?what is wrong with having a sample contaminated by genomic DNA?how does that affect transformation?

    thank you.

    1. Avatar of m v m v says:

      i dont think the editor or contributor is responding to anymore questions…did u run a gel? it might interfer with the quantification of the target by smearing on a gel. gnomic dna is long and can interfer wit hthe effectiveness of the target…better yet broken gnomic, nicked or open circular dna can decrease your overall percentage of supercoiled or plasmid.

    2. Avatar of Nick Oswald Nick Oswald says:

      Hi Pijaw

      gDNA contamination should not affect your transformation since none of the genomic pieces should be transformable. gDNA WOULD be a problem if you wanted to use your plasmid for anything PCR-based (since the gDNA would be non-specifically amplified). It would also be a problem if you wanted cut a piece of your plasmid using restriction enzymes, since the enzyme would cut in the genome, creating sticky ends that would interfere with downstream applications.

  10. Avatar of m v m v says:

    better yet does anyone have a solid recommendation for a metering pump that can be programable without an rs cable or any other interface…just a stand alone pump?
    thanks

  11. Avatar of amitavagts amitavagts says:

    hi has anyone tried isolation of plasmid dna by alkaline lysis method from mammalian cells transfected with plasmid of interest

    1. Avatar of abo177 abo177 says:

      Hi Dr Suzanne,
      I am extracting plasmid DNA from transconjugates of Mupirocin High level resistant staphylococcus aureus, i use CTAB lysis and i use coloform/isopropanol to extract the plasmid DNA.
      My problem is that i am getting smears recently for no reason, I am following all precautions as written in almost all protocols.
      I am getting all figures of smears, upper tailed, global smear, or lower tailed..I am doubting the presence of either genomic DNA, or nucleases at my final prep. so shall i clean up the final TE- plasmid solution by boiling or what do u suggest?

      Thanks
      Ahmed

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