Traces with delayed sequencing signal start
Identification of traces with a delayed signal start
- The first signal peak in the raw channel is found after scan 3200 for 50cm traces and scan 2700 for 36cm traces. Traces normally start between scan 1800 and 2200 for 50cm traces and scan 1500 and 1900 for 36cm traces.
- The signal in the processed channels is often of low resolution with wide peaks.
Figure 1. Example of a good (A) and a delayed start (B) 50cm trace. The raw channel is shown.
Causes of delayed signal start sequencing traces
- Capillary overload. Unlike a lot of DNA sequencing problems delayed peak signal has only one cause - capillary overload. The two most common causes of capillary overload are too much template DNA or dirty template DNA contaminated with proteins an/or salt.
Solving capillary overload problems
- Use less template DNA. Modern ABI capillary sequencers require far less template DNA than the old slab based sequencers, however, many researchers are still using protocols developed for the slab based system. Try the suggestions made in the DNA sequencing protocol tips page.
- Use cleaner DNA. Try changing you template preparation protocol. A good cheap template preparation protocol that works well with sequencing is the ExoI/SAP clean up.
Return to the main DNA sequencing troubleshooting page.