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. POP7 polymer traces run at 8.5kV normally start between scans 1800 and 2200 for 50cm traces and scans 1500 and 1900 for 36cm traces.
- The signal in the processed channels is often of low resolution with very broad peaks.
Figure 1. Example of a normal raw channel
Figure 2. Example of late dye signal
Figure 3. Example of late dye signal.
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 templates DNA contaminated with large amounts of proteins and/or salts.
Solving capillary overload problems
- Use less template DNA. Modern ABI capillary sequencers require far less template DNA than the old slab based sequencers (i.e. ABI 377) , however, many researchers are still using protocols developed for the slab based system. Try the suggestions made in the DNA sequencing protocol tips page for ways to improve.
- Use cleaner DNA. Try changing your template preparation protocol. A good cheap template preparation protocol that works well with sequencing is the ExoI/SAP clean up of PCR products. Try different kits as some work better than others for DNA sequencing.
Return to the main DNA sequencing troubleshooting page.