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.
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….
I think we all have been through those my-PCR-product-didn’t-get-amplified days. Sometimes, playing around a bit more with the PCR-conditions brings luck, or sometimes it doesn’t work at all. These days we have access to many different types of DNA polymerases, ultrapure and buffered nucleoside triphosphates, and other necessary starting materials in convenient concentrations; but still […]
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