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To isolate plasmid DNA, you crack your cells open and perform a miniprep, trying hard to avoid contaminating genomic DNA. For genomic DNA, you crack your cells open in a different way and try to isolate as much of the contents as possible.
So what’s the difference in the protocols?
In this article, we will look at plasmid and genomic DNA extraction, and the ways in which these techniques differ.
Genomic DNA Extraction:
1. Lysis: Just Crack Them Open
Genomic DNA (gDNA) extraction is the simpler procedure because strong lysis is the only step necessary to release gDNA into solution. For yeast, plants, and bacteria, lysis involves enzymatically breaking the strong, rigid cell wall before mechanically disrupting the plasma membrane.
Cell walls are usually digestible with lysozyme, which hydrolyzes cell wall peptidoglycans, and the serine protease proteinase K. For certain gram-positive species, lysostaphin will further aid enzymatic digestion. You may need to use different enzymes for more exotic species with different cell wall compositions.
Mechanical cell wall disruption represents a more universal lysis method for gDNA extraction. Bead beating is popular, and you can easily do this 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.
For tough filamentous fungi (e.g. Aspergillus and Fusarium spp.), cellular material is often snap-frozen in liquid nitrogen and milled in a pestle and mortar followed by rapid vortexing in solution with an appropriate lysis buffer.
2. … and purify
Following cell lysis (which brings the gDNA into solution), the only thing left to do is to purify the sample. You can do this by using phenol-chloroform or spin filter membrane technology with added guanidine salts that promote binding to silica.
3. A few words of advice
Chromosomes will break during purification because they are simply too large to stay intact; for most applications this is not an issue. Indeed, for PCR and qPCR, the breakage may actually be advantageous because it aids DNA melting, resulting in more efficient amplification reactions. However, for applications using large DNA fragments (e.g. long read sequencing and long range sequencing), this may be a serious issue. If you do need to isolate ultra-long DNA fragments for these applications, you should consider another setup.
The E.coli chromosome is just over 4.5 MB in size, amounting to approximately .005 picograms per cell. A typical overnight culture from a single starting colony will contain approximately 1-2×109 cells/ml. Theoretically, that means that 1 ml of culture should yield about 5 µg of gDNA per 109 bacterial cells. Take this into account when calculating how much DNA you need for your chosen application.
Plasmid DNA Extraction
Plasmid DNA extraction is a bit trickier because plasmid DNA must be kept separate from gDNA. This separation is based on size, and good separation relies on using the right 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 enzymes or bashing it with glass beads. Birnboim and Doly invented the (virtually) universal method for plasmid DNA extraction via alkaline lysis in 1979.
The lysis buffer contains sodium hydroxide and SDS, which completely denature plasmid and gDNA (i.e. separating the DNA into single strands). It is critical that this step is performed quickly because excessive denaturing may result in irreversibly denatured plasmid.
Next, the sample is neutralized in a potassium acetate solution to renature the plasmid. This is key to the separation of plasmid and gDNA. Because plasmids are small, they can easily reanneal forming dsDNA. Genomic DNA, however, is too long to reanneal fully, and instead it tends to tangle so that complimentary strands remain separated.
During centrifugation, gDNA (bound to protein) forms a pellet while plasmid DNA remains soluble. It is key at this step not to vortex or mix the sample vigorously because gDNA breaks easily, and broken gDNA may be small enough to reanneal and go into solution with the plasmid.
Plasmid DNA in the supernatant can then be ethanol precipitated or cleaned up using phenol-chloroform or a spin filter. If you are using spin filter technology, the neutralization buffer will contain guanidine salts so the lysate can bind the silica directly for further washing and elution. The resulting DNA is pure enough for most downstream molecular biology applications. If you need the plasmid for transfection, anion-exchange purification is a better choice to remove contaminating endotoxin. Endotoxin removal is also possible using faster silica-based purification setups.
This method is compatible with mammalian and other eukaryotic plasmids as well as other small extra-chromosomal DNA species. Bear in mind, however, that plasmid copy number is often much lower in mammalian cells and plant extranuclear organelles.
3. … and some words of advice
Plasmid DNA is typically 3-5 kb, depending on insert size. The specific origin of replication present will influence the plasmid copy number per cell. A typical high copy plasmid such as pUC or pBluescript should yield 4-5 µg of DNA per ml of culture.
To isolate high yields of plasmid DNA, use cultures in late log phase or early stationary phase. Prepare cultures using fresh single colonies and fresh selection antibiotic at the right concentration for plasmid maintenance. It is important not to overgrow bacterial cultures as this may result in gDNA contamination in the plasmid extract.
Nowadays, there is a kit for everything, and the internet contains vast information about extracting DNA from plasmids, genomes and everything in between!
Maximizing DNA Yield From Whole Blood. https://bitesizebio.com/30243/maximizing-yield-whole-blood-dna-extraction/
Originally published in 2014, republished in 2019.