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11024 09.06 Sanger Quick Tips Banner
QUICK TIPS GUIDE

Sanger Sequencing Quick Tips Guide

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 100.

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.

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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 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 2: Purifying a Single-band PCR Product

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

 

 

Volume 4: PCR Visualization by Gel Electrophoresis

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