We cut a slice of the target gene from the gel which ran electrophoresis. And then purified the gene from the gel by using Gel-M column, WN buffer and WS buffer. After these steps, we ran another electrophoresis of the purified gene and there was no band showed on the picture. What might happened to make this situation happened?
There could be several reasons why no band showed on the picture after running another electrophoresis of the purified gene. Here are a few possibilities:
1. Insufficient elution: During the purification process using the Gel-M column, it is essential to ensure that the target gene is adequately eluted from the gel. If the elution process was not optimal, it can result in low recovery or loss of the gene, leading to no band on the subsequent electrophoresis gel.
To improve the elution process, you can try increasing the incubation time, gently shaking the column, or adjusting the elution buffer composition or pH according to the manufacturer's instructions.
2. Enzyme inactivation: Gel purification often involves the use of enzymes to digest the gel and release the target gene. If the enzymes used for gel digestion were not fully active or if the digestion conditions were not optimized, it can result in incomplete digestion, incomplete release, or degradation of the target gene.
To address this, ensure that the enzymes used are fresh, properly stored, and within their specified activity period. Optimize digestion conditions such as enzyme concentration, digestion time, and temperature based on the manufacturer's recommendations or previously established protocols.
3. Sample loss during handling: It is crucial to handle the purified gene sample carefully to prevent any loss or contamination. If the sample was accidentally lost or spilled during the handling process, there will be no DNA to detect on the gel.
To prevent sample loss, use proper pipetting techniques, ensure a secure seal on the columns or tubes used for purification, and work in a clean and organized environment.
4. Transfection or transformation inefficiency: If the goal is to use the purified gene for transfection or transformation, the absence of a band on the gel could suggest that the purification process did not yield sufficient amounts of the gene for successful transfection or transformation. In such cases, even if there is some DNA present, it might be below the detection limit of the gel electrophoresis.
To address this, consider optimizing the purification process to improve the yield of the target gene. This could include adjusting the starting amount of DNA, optimizing the purification protocol, or considering alternative purification methods.
It's important to carefully analyze each step of the procedure to identify potential areas for improvement and troubleshoot accordingly. Additionally, consulting relevant literature or seeking guidance from experienced researchers in your field can provide additional insights and troubleshooting strategies.