Why electrophoresis fail

Estimation by gel is difficult. If you are not experienced at this, find someone who is more experienced to help you. Double-check all calculations!!!

There is a six-orders-of-magnitude difference between the two! These signal-strength numbers are an indication of the relative fluorescent strength of the bands in your lane. We typically examine just the G signal to simplify comparisons. Good samples will have a G signal of If your signal strength is below , background bands that are normally too low to see will become very evident and will interfere with base-calling.

If your signal is below about 80, your peaks may get lost in the background noise, and we will report only a blank lane.

The possible causes are almost the same as those for weak bands. You might get lucky sometimes. Baseline noise varies from lane to lane, run to run, and instrument-to-instrument. If you happen to get a low-noise situation, even a sample with a G signal of 50 will give great sequence. The next time, that exact same sample will bomb, because the noise was too high.

If you see this, you usually have two sequences superimposed on each other. There are several common causes:. A similar outcome is often seen in which the bands start out fine, but later on become superimposed.

This is described further down this page. Another example, this time with templates that might be related. Note the alignment of the peaks:. Below is an example actually, a fairly mild example of a ski-slope :. Capillary electrophoresis instruments such as our ABI Model sequencers are quite sensitive to the presence of excess salt.

It tends to favor detection of smaller fragments over larger ones. Our purification protocols are designed to minimize this problem, but it still occurs at times. The terminator concentrations are carefully adjusted to statistically favor long extension, and the enzyme is modified to be able to accept bulky dye molecules as substrates. This is a common artifact of automated sequencing that arises from complexes formed between the sequencing dyes and unknown other components often contaminants.

There are two things that cause this artifact:. First, if our sample cleanup is flawed, we might have left excess unincorporated dyes in the sample. Second, your sample itself may have a contaminant that binds unincorporated dyes. The largest peak was always green, and this only happened in the first ca. NOTE: We have not seen this artifact since changing to a newer type of dye, in early This section being kept primarily as a courtesy to clients of other Cores that still use these older dyes.

Secondary structure in the template is the most likely cause of this problem. The polymerase is presumably unable to progress through some stem-loop form. These will almost always exhibit strong sec-structure effects. Band intensities are much less variable with these newest dyes.

The concentration of the gel must also be correct to avoid errors. If the concentration is too high or too low, the fragments will migrate either too slowly or too quickly. This will lead to errors in resolving the different bands. During the electrophoresis run, care must be taken to ensure that the voltage is steady.

Any fluctuations in the voltage will result in unsteady migration of DNA fragments, leading to errors in reading the bands. The buffer solution must also be of the correct composition, as a buffer with the wrong pH or ionic concentration will change the shape of the DNA fragments, also changing their migration times.

Most importantly, the gel must be visualized properly. If the concentration of the dye or radioactive probe used to visualize the samples is too high, the resulting image will be very messy, as residual fragments will also be visualized.

If the gel concentration is too low, there will be no visualization. When the correct processes have been followed during all stages, gel electrophoresis will yield results that are accurate and can be used with great confidence.

As with all scientific procedures, gel electrophoresis can be prone to errors, but these can be minimized with proper preparation and handling. Joshua Suico is a university teacher specializing in chemistry and the life sciences. He holds a Master of Science degree in chemistry. If your thermal cycler does not have a heated lid, overlay the PCR reaction with wax.

Make sure students close the lid of the PCR tube properly. Ensure that the electrophoresis buffer was correctly diluted. Make sure that the solution is completely clear of "clumps" and glassy granules before pouring gels.

The gel was not stained properly. Repeat staining. Malfunctioning electrophoresis unit or power source. Contact the manufacturer of the electrophoresis unit or power source.