Isolating genomic and plasmid DNA for further investigation and downstream application (e.g. PCR, sequencing, etc.) requires totally different protocols. While isolating genomic DNA merely requires you to crack open the cell walls and purify the resulting sample, extracting plasmid DNA may be a bit trickier and more complicated than this. Here’s a rundown on how these techniques differ.
As mentioned earlier, extracting genomic DNA is a simple affair. All you need to do is disrupt the cell wall using lysozome (an enzyme that effectively hydrolyzes the peptidoglycan component of the cell wall) and proteinase K (a serine protease used in digesting proteins and removing contaminants). You may also use the antibacterial enzyme lysostaphin when working with certain gram-positive species to further enhance enzymatic digestion.
In the same manner, samples may also be subjected to physical and/or mechanical methods to release the genomic DNA from the cell lysate. Most researchers consider bead beating as the lysis method of choice since it is generally faster and more thorough than enzymatic lysis. However, for tough filamentous fungi such as Aspergillus and Fusarium, the cellular materials are best extracted by freezing the sample in liquid nitrogen before using other physical or mechanical methods.
Once the genomic DNA has been successfully extracted, a phenol-chloroform mixture or a suitable protease is used to remove the lipid membranes from the solution. The unwanted contaminants can also be removed by precipitating them with sodium or ammonium acetate.
Some Tips to Consider:
- Genomic DNA can be easily degraded or corrupted. To prevent gDNA from degrading quickly, remember to keep samples at low temperatures, use chemical inhibitors to block DNase activity, and perform a protein precipitation step to completely remove the nucleases in the lysate.
- Since DNA chromosomes tend to break during the purification process, you may need to choose a more appropriate setup such as MegaLong™ if your downstream application calls for extra long DNA fragments.
- Don’t forget to calculate how much DNA you need for your application. Keep in mind that every 1 ml of culture yields ~5 µg of gDNA per 109 bacterial cells.
While gDNA extraction is pretty straightforward, extracting plasmid DNA can be a little more complicated since you should be able to identify and use the appropriate lysis method to successfully separate the plasmid DNA from the gDNA. Basically, a milder treatment (i.e. alkaline lysis) is required when extracting plasmid DNA. Here’s how you go about extracting them.
The procedure starts with the cultivation of bacterial cells in varying amounts of growth medium. When sufficient growth is achieved, you can remove the cells from the medium through centrifugation.
Resuspension and Cell Lysis
A cell pellet from the saturated culture is resuspended in an isotonic solution containing Tris, EDTA (to disrupt the cell wall and prevent DNases from damaging the plasmid), glucose (to prevent the cells from bursting) and RNase A (to degrade cellular RNA during cell lysis). An alkaline solution containing sodium dodecyl sulfate (SDS) is then added to facilitate cell lysis and the complete denaturation of both genomic and plasmid DNA along with all the proteins in the solution.
Neutralization, Cleaning and Concentration
A potassium acetate solution is then used to neutralize the sample and separate the plasmid DNA from the gDNA. The smaller plasmid DNA tends to renature easily while the larger, more complicated gDNA remains denatured and precipitates out of the solution.
Upon centrifugation, gDNA will form a pellet while plasmid DNA remains soluble. The plasmid DNA remaining in the supernatant can then be precipitated with ethanol or purified using a phenol-chloroform mixture or spin filter technology.
Some Tips to Consider:
- The cell lysis step should be done quickly since overdoing it can irreversibly denature the plasmid.
- While the resuspension and lysis buffers should be mixed thoroughly, make sure you do not vortex or mix it vigorously to prevent the DNA from breaking into smaller fragments. If they are small enough, broken gDNA can reanneal and remain in the solution.
- Wear gloves and appropriate eye protection when working with harsh chemicals such as NaOH and SDS.