Displayed below is the difference in ladder peak height using formamide with low conductivity (upper electropheragram) and data using formamide with high conductivity (lower electropheragram).
There is a large difference in the peak height detection between the two samples (the highlighted allele in the top panel is 898 relative fluorescence units (RFUs) vs. 122 RFUs for the same allele in the presence of formamide with high conductivity). Note that formate ions are not fluorescently labeled and no fluorescence is detected with the electrokinetic injection of these by-products.
Electrokinetic injection does not discriminate between charged DNA fragments and formate ions; both of these charged particles compete for injection. In the presence of formate ions, the result is fewer DNA fragments being injected into the capillary due to competition. As shown in the figure above, the effect of preferential formate ion injection can be extremely detrimental. In addition, signal can be decreased for samples with high salt concentrations. By adding a very small amount of amplified product in the presence of formamide, the salts in a sample are very dilute.
Resolution is another means to evaluate the quality of formamide. The resolution is a measure of how well the peaks are separated. The overall performance of the capillary and all reagents used in the electrophoresis can be assessed by monitoring resolution.02, 03 In order to make discrete allele calls, the system must be able to resolve to the base pair. Low quality formamide can prevent this single base pair resolution.
There is no longer the need for laboratories to conduct quality control testing when using ultra-pure products such as HiDi™ formamide (Applied Biosystems). For example, this product, has stabilizers present to maintain a low measured conductivity of 5 µS/cm.
High quality formamide should:
- Be greater than 99% pure
- Have a minimum conductivity of 100 µS/cm
- Have low water content
- Be packed under inert gas
Continued exposure to air and subjection to multiple freeze-thaw cycles can cause the formamide to breakdown. It is suggested that aliquots of formamide that is stored in frost-free freezers be placed in thermal ice blocks to protect from temperature cycling.
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