G Dye Peaks at Base 190 and 400

Late "G" dye peaks at base positions 190 and 400

Identification of late "G" dye blobs problems

• One or more large "G" channel peaks in the trace regions around base 200 and 400.
• The earlier of the two "G" peaks is normally the largest. In some cases the ~400 base G peak is not visible at all.

Figure 1. Late "G" dye blob in the base 180 - 200 region.

Figure 2. Late "G" dye blob in the base 400 region.

Causes of late "G" dye peaks

• The cause of late "G" dye peaks is thought to be due to the breakdown of the BigDye sequencing chemistry before sample clean up (ie before the leftover labeled nucleotides are removed).
• The major cause of breakdown of the BigDye chemistry is excess heating or high numbers of freeze/ thaw cycles.
• The problem of late G peaks is often only detected if the sample clean up is not performed correctly. In other words, late G dye blobs tend to only be observed when the trace contains early dye blobs.

Solving late "G" dye peak problems

• Avoid overheating the DNA sequencing reactions. Try and keep the denaturation temperature in the sequencing thermocycling below 96˚C and under 20 seconds.
• Avoid excessive numbers of freeze/thaw cycles of the BigDye chemistry. It is a good idea is to aliquot out the BigDye stock into small (10 - 20 µl) lots to minimize the number of time the sequencing chemistry is defrosted.
• If the trace contains early dye blobs then check and/or modify your sequencing clean-up protocol. You may want to try using the phenol/butanol sequencing clean-up protocol.

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