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Latest Volume: Sanger Quick Tips 5

Tips and tricks for troubleshooting a poor sequencing reaction from a PCR template:

  • Failed reactions (i.e., no priming)
     Usually indicated by a chromatogram with a signal intensity of
  • Nonspecific reactions
     Characterized by two or more overlapping traces in the
     chromatogram that represent different populations of
     sequencing products.
  • Spectral pull-up
     Occurs when the signal intensity of the sequencing reaction is so
     strong that the fluorescence signal effectively spills over into the
     other collection channels.

Download Quick Tips

The Sanger Quick Tips Series Also Includes:

Volume 1: Producing a Single-band PCR Product

  • PCR primer design tips and guideline
  • Polymerase enzyme selection pros and cons
  • Buffer and cycling parameters
  • PCR adjunct selection

Volume 2: Purifying a Single-band PCR Product

  • Enzymatic PCR clean-up
  • Purification by DNA-binding matrix

Volume 3: Purifying a Non-specific PCR Product

  • Isolation of a single-band by gel purification 
  • Single-band recovery using band-stab PCR
  • Purifying a single product using nested primers

Volume 4: PCR Visualization by Gel Electrophoresis

  • Running buffer selection
  • Voltage and run-time optimization
  • Loading dyes
  • Target agarose percentages
  • Troubleshooting common gel problems