Home FAQs Why does my Lambda DNA molecular weight marker appear smeared and not represent the correct banding pattern?

FAQ: Why does my Lambda DNA molecular weight marker appear smeared and not represent the correct banding pattern?

These banding aberrations are almost always a result of the preparation of the DNA molecular weight marker for electrophoresis. For example, the Lambda DNA markers (Hind III and BstE II digests) require a brief heating before electrophoresis (we recommend 3 minutes at 60C, followed by placing on ice or loading directly onto a gel) to dissociate the 12-base sticky ends of the cohesive ends. Without this heating, the 23kb band and the 4.3kb band of Lambda-Hind III anneal by H-bonding between the complementary bases to form a 27kb band. Visually, this results in a decrease in intensity of the 4.3kb band. Without heating lambda DNA-BstE II, the 8.4kb band and the 5.6kb band anneal to form a 14kb band. Visually, this results in a decrease in intensity of the 8.4 and the 5.6kb bands plus the appearance of a 14kb band.

One caveat to heating DNA markers is that double stranded DNA requires a minimal amount of ionic strength to keep it from denaturing. This denaturing of strands may result in irregular or fuzzy banding patterns. Therefore, we do not recommend diluting DNA markers in water, but rather in a buffer with minimal ionic strength such as TE buffer.