No band in UREA-PAGE with labeled oligo?

If you are not seeing any bands on a urea-polyacrylamide gel electrophoresis (PAGE) when visualizing labeled oligonucleotides, changing the excitation wavelength may not be the primary solution. Instead, consider the following factors that could be contributing to the lack of visible bands:

Gel composition: Ensure that the urea-PAGE gel you are using is properly prepared. Urea-PAGE gels typically require higher urea concentrations and lower acrylamide concentrations compared to regular denaturing PAGE gels. Verify that the gel composition and concentrations are appropriate for your specific oligonucleotide size range.

Denaturation of samples: Ensure that your oligonucleotide samples are sufficiently denatured before loading onto the gel. Heat your samples at 95°C for a few minutes and quickly cool them on ice before loading. This step ensures that the oligonucleotides are in a single-stranded form for accurate separation.

Loading buffer: Use an appropriate loading buffer that contains denaturing agents such as formamide or EDTA to maintain single-stranded conformation during electrophoresis. The loading buffer should also contain a tracking dye for visualization purposes. Ensure that you add the loading buffer to your samples before loading them onto the gel.

Electrophoresis conditions: Optimize the electrophoresis conditions, such as the voltage, run time, and buffer composition. Urea-PAGE typically requires a lower voltage and longer run times compared to regular PAGE. Adjust these parameters accordingly to allow sufficient migration and separation of the oligonucleotides.

Detection method: Consider the detection method you are using to visualize the labeled oligonucleotides. If you are using a fluorescent label, such as a fluorescent dye or a radioactive label, ensure that your imaging system is set up to detect the appropriate excitation and emission wavelengths for the specific label you are using. Check the specifications of your imaging system and adjust the settings accordingly.

Labeling efficiency: Assess the efficiency of your oligonucleotide labeling reaction. If the labeling efficiency is low, it may result in weak or undetectable bands. Verify the labeling protocol you are using, including the labeling reagents, reaction conditions, and purification steps. Consider optimizing the labeling reaction to improve the labeling efficiency.

Before changing the excitation wavelength, ensure that you have exhausted other troubleshooting steps mentioned above. Changing the excitation wavelength is generally not the first approach unless you have specific reasons to suspect that the excitation wavelength is incompatible with your labeled oligonucleotide or imaging system.

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