Troubleshooting Your Samples

Problems Associated with Primers

Poor Priming Resulting in Very Weak Signal

  • Melting temperature too low: low G/C content or primer too short
  • Secondary structure of primer, particularly at the 3' end
  • Secondary structure of template in region of hybridization

Adequate Signal Strength but Data is Noisy

  • Secondary hybridization site; generally will see too many peaks (peaks under peaks).
  • More stringent annealing conditions can eliminate this.
  • Impure primer; may see a "shadow sequence".

Incorrect Primer Sequence

  • Mismatch on 3' end will generally result in completely flat line or no signal

Problems Associated with DNA Templates

Poor Template Quality

  • Impurities such as protein, RNA, ethanol, salts, etc. in template DNA will produce high background and pour quality data. Please refer to the chart above regarding allowable contaminant concentrations.

Amount of Template DNA

  • Not enough template—Results in low signals, noisy data, errors in base calling.
  • Too much template—Peaks in sequencing data show trailing and may be accompanied by a weaker signal.

G/C Rich Templates

  • Are generally more difficult to sequence.
  • Overall weak signal results in noisy data—presumably this is related to the tighter duplex and greater difficulty in denaturing the template, as well as the presence of secondary structure.

Template Secondary Structure

  • Inverted repeats for example give good signal in the beginning but then signal drops off suddenly.

Questions?

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