DNA plasmid isolation is integral to biology as it’s used in many functions. This includes DNA sequencing, cloning, in vitro translation, gene therapy, blotting, and more. However, while the process of DNA plasmid isolation is a crucial aspect of biology, it also comes with its challenges.
In plasmid isolation, it is up to the technician to use the correct lysis method to correctly extract the DNA. in most cases of DNA plasmid isolation, alkaline lysis is preferred, as it is mild. Alkaline lysis isolates plasmid DNA and proteins by breaking cells apart. Then the proteins are removed via alcohol precipitation. Read on to learn more about the processes of plasmid isolation from DNA and its challenges.
The Process of Extracting Plasmid DNA
The first step in DNA plasma isolation is cultivating bacterial samples, followed by colony picking cell culture. Picking colonies from agar plates are the norm for typical colony picking. However, this can be a tedious process, and more and more laboratories are turning toward automated colony pickers. Some automated systems can pick up to 3,000 colonies an hour, which is extremely cost-effective for a laboratory.
After bacterial samples are cultured and picked, the cells (which are now pelleted), are placed into a buffer solution. Once this process is complete, it’s time to lyse the cells. Place the cells in an alkaline solution, such as sodium hydroxide or sodium dodecyl sulfate (SDS). These alkaline solutions break down the cell membranes and convert double-stranded DNAs (dsDNA) to single-stranded DNAs (ssDNA) as part of plasmid isolation using DNA.
Next, in DNA plasmid isolation, potassium acetate solution is used to separate the plasmid DNA from the genomic DNA (gDNA). After centrifugation, gDNA remains soluble. Next, you precipitate the plasmid DNA with ethanol (ETOH). Leave the sample for 10 minutes so that the alcohol can evaporate. The DNA pellet must also be resuspended in another buffer solution containing Tris, RNases, and EDTA).
What Are the Challenges of DNA Plasmid Isolation?
While this may sound like a simple process, it is quite tedious, and there is no room for error. That requires highly skilled and trained scientists and technicians in the lab to help foster perfection.
One downside of picking bacterial colonies by hand is that it is time-consuming and slows the process of DNA plasmid isolation. Automated colony pickers can be quite useful and help but a big slash in your labor costs.
There are other common mistakes when it comes to DNA plasma isolation. These include:
- Cell lysis must be performed quickly. Otherwise, the plasmid can be denatured and you’ll have to start over.
- Lysis buffers and resuspension must be thoroughly mixed but not too vigorously. This prevents DNA from breaking into smaller fragments. Small gDNA can break and stay in the solution.
- NaOH and SDS are caustic, so it’s important to always wear eye and clothing protection when working with these chemicals. Generally speaking, it should be a custom every time you work in the lab.
Hudson Robotics has been the leading supplier of automated products for over 38 years. To learn more about DNA plasmid isolation, speak to a representative today!