Multiple Peaks/Mixed Signal
- Electropherogram has two or more sequence peaks at the same location. The secondary peaks may be the same height as the primary peaks down to about 20%. Lower levels of mixed signal (below 20%) are normally base- called well by the KB
- The trace signal strength is good (>200U) in the raw channel
- The quality scores are low with often less than 100 Q20+ bases
- The base called sequence does not match the expected sequence or any known sequence in GenBank
- Two or more templates were present in the reaction. This is the most common cause of mixed template
- A "double pick" of two colonies. This can occur when the colonies are too close together on the colony plate
- Two primers were present in the sequencing reactions. This can occur when using premixed PCR reagents for sequencing where the primer stock is actually a mix of universal forward and reverse oligonucleotide primers
- The PCR fragment was not purified of leftover primers before sequencing
- Two priming sites are present in DNA template. This can occur when a PCR product with universal priming site tails is cloned into a plasmid
- Poor quality PCR template containing multiple DNA fragments was used
- Too low a primer annealing temperature was used in the sequencing reaction
- Different sequencing reactions were accidentally mixed at the clean-up stage. This can also sometimes occur if the same tip is used without rinsing
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- Make sure only one DNA template is present. Prepare a new plasmid prep making sure that only one colony is selected. If sequencing a PCR product check that only one PCR product is present by running an agarose gel.
- Check the template for possible multiple priming sites. This can often occur when a fragment containing the priming site is sub-cloned into a vector that also contains the priming site. If two sites are present use a different primer. Be very careful of this problem when using the M13 universal primers.
- Ensure that you use a PCR clean-up protocol that removes leftover PCR primers.
- Check the predicted melting temperature of the sequencing primer. If it is more than 5ºC above the annealing temperature used in the sequencing reaction (50ºC) then raise the annealing temperature. Because of the inclusion dITP in the BigDye sequencing mix the annealing/extension temperature can't be raised above 60ºC. If your primer still misanneals at 60ºC then synthesize a new primer.