I'm attempting to separate two DNA oligos of length 120bp and 100bp. I've been running them with 2x formamide loading buffer for 3 hours at 65V. I understand constant power is important for the temperature of the running buffer to help denaturation. My lab only has a constant Volts or Amperes power supply, so this is what I've been using. I've been considering purchasing a more advanced power supply that has constant power. How important is this for overall denaturing PAGE settings? Is there anything wrong with my current settings? My current method semi-works and I'm considering continued troubleshooting or exploring constant power.
Hello Isabel Bravo ,
If you are using the power supply in the constant voltage mode, say, and the current increases with time, the power dissipated in the gel increases with time and this should hasten the denaturing by increasing the temperature in the gel. By this I mean, if the macromolecule denatures at 35°C, then heating it beyond this temperature will not cause it to refold to its biologically active configuration. However, if the current decreases signicantly, and quickly, the gel's temperature may not reach the denaturing temperature.
Of course, it would help if you knew the minimum denaturing temperature of the macromolecule, and you could safely measure the temperature of the gel. Since you are probably using fairly high voltages to perform the electrophoresis, you probably only have access to the outer surface of the tubes in which you cast the gels. Be very careful of leakage currents on the outer surfaces of these tubes. Is your power supply a linear or a switching supply?
Regards,
Tom Cuff
maintaining constant power is generally preferred over constant voltage. Constant power ensures a more consistent rate of heating throughout the gel, promoting uniform denaturation of nucleic acid samples. This is particularly important when dealing with DNA fragments that require denaturation.
In constant voltage electrophoresis, as the resistance of the gel changes with time due to temperature variations, the power fluctuates. This can result in uneven denaturation and separation of DNA fragments, especially when dealing with samples of different lengths.
Given your current setup with a constant voltage power supply, there are a few considerations:
Gel Composition: The gel composition, including the percentage of acrylamide, the concentration of denaturing agents (such as urea), and the formamide loading buffer, plays a crucial role in denaturation. Ensure that the gel composition is appropriate for your target DNA fragments.
Run Time: Running your samples for 3 hours at 65V suggests an extended run time, which may be contributing to the separation of your DNA fragments. You may want to optimize the run time based on the expected size difference between your 120bp and 100bp fragments.
Temperature Control: Although constant power is preferable, temperature control is critical. Ensure that the temperature of the running buffer is maintained within the optimal range for denaturation. Formamide loading buffer is often used to assist in denaturation by disrupting hydrogen bonds.
Power Supply Upgrade: If possible, consider investing in a power supply that allows for constant power settings. This can enhance the reproducibility and efficiency of your denaturing PAGE.
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