Sanger Sequencing Quick Tips Guide
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