There are several methods used in life science laboratories to break open cells (a.k.a. achieve cell lysis) and access the materials within (e.g. DNA, proteins, organelles, proteins, DNA, mRNA, etc.). One such method is alkaline lysis.
What is Alkaline Lysis?
Introduced in 1979, alkaline lysis is a method used to isolate plasmid DNA from relatively small volumes of bacteria. There are essentially 5 steps in the alkaline lysis process.
Step 1: Cell Growth & Harvesting
Researchers begin by growing and harvesting the bacterial cell culture containing the plasmid DNA. Once growth is sufficient, centrifugation is used to remove them from the growth medium (this process is called pelleting the cells).
Step 2: Re-Suspending Pellet
Next, the pelleted cells are re-suspended in a solution comprised of EDTA, RNas A, Tris and glucose. This solution is often referred to as “Solution 1”.
Step 3: Cell Lysis
Then, the lysis buffer is mixed with the re-suspension solution. The lysis buffer is often referred to as “Solution 2”, and is comprised of sodium hydroxide (NaOH) and Sodium Dodecyl (lauryl) Sulfate (SDS). The NaOH breaks down the cell wall, disrupts hydrogen bonding among DNA basis, and triggers denaturation, which is when gDNA is converted to dsDNA. The SDS also aids in the denaturation process.
Step 4: Neutralization
After cell lysis takes place, potassium acetate – which is often referred to as “Solution 3” is added. This decreases alkalinity, and allows ssDNA to re-nature to dsDNA.
Step 5: Cleaning & Concentrating
Finally, it is necessary to clean the solution and concentrate the plasmid DNA. A common way to do this is via phenol-chloroform extraction, followed-up with ethanol precipitation and affinity chromatography-based methods. Other approaches include ethanol precipitation, silica column-based kits, strataclean resin, and magnetic beads.
Limitations of Alkaline Lysis
There are a key drawbacks associated with alkaline lysis that researchers must be mindful of in order to ensure that their efforts are both productive and cost-effective. These drawbacks include:
- Contamination: If the Alkaline lysis process is too rigorous, then the gDNA will shear and produce shorter stretches. This can re-anneal and contaminate the plasmid preparation.
- Inefficiency: Alkaline lysis can be very time consuming and difficult to automate. As such, it may not be viable or cost-effective.
- Yields: Alkaline lysis often results in low and variable yields; particularly when using multiple templates, or when using a variety of hosts or vectors. It is therefore difficult to establish a high-throughout sequencing pipeline.
Pion High Pressure Homogenizers
Pion's High Pressure Homogenizers are designed to help researchers avoid the limitations and drawbacks of alkaline lysis. Specifically, the equipment is designed to accommodate different cell disruption strategies, deliver consistently high yields in the least amount of time, help prevent contamination reducing the likelihood of shearing gDNA, and support researchers as they scale from small samples to larger clinical trials.
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