To reduce the effect of impurities in a DNA sample, you can use less sample DNA (minimum of 50 ng) or perform an extra DNA purification step.
Impurities in extracted DNA can affect all or some MLPA probes in a probemix, which can lead to unexpected results. This is mainly a problem when impurities are present in high concentrations, or when concentrations differ between samples within an experiment.
If your sample DNA is sufficiently concentrated, you can reduce the amount of sample DNA that you add to the MLPA reaction (minimum of 50 ng DNA). This reduces the concentration of impurities carried over from the DNA sample into the MLPA reaction.
|Methods to determine DNA concentrations may over- or underestimate the concentration. To ensure that you used enough DNA for reliable results, evaluate the median peak height of the four Q-fragments at 64, 70, 76 and 82 nt. As long as this stays below 33% of the height of the 92-nt benchmark fragment, sufficient DNA was used. Read more about Q-fragments in this article.|
If using less DNA is not an option, or does not improve results, you can perform an extra DNA purification step. Examples of methods that you can use include ethanol precipitation, or silica-based columns.