Overlapping peaks in all or part of the sequence

 

Multiple sequences-electropherogram-website

Overlapping peaks, with the same or different heights.

 

 

Multiple sequences-raw-website

Raw data trace displays signal intensity in the acceptable range, 100-4,000 RFUs, indicating that the double peaks are not due to weak signal.

 

 

Possible causes

Recommendations

Mixed plasmid preparation

Re-isolate the DNA from a single colony and re-sequence.

Multiple PCR products

Check the PCR template on gel for single band.

PCR template not cleaned up (residual PCR primers amplified)

Make sure the PCR primers and dNTPs have been removed.

Multiple priming sites

Make sure the sequencing primer has only one binding site.

Frame shift mutation

Use a primer after the mutation or sequence the complementary strand. If a PCR product, clone and re-sequence.

Primer with n-1 contamination (poor purification during synthesis can lead to a mixture of full length and n-1 primer, which gives a shadow sequence, one base behind)

Order new sequencing primer, preferably HPLC purified.