Troubleshooting
Poor Quality Template
Poor quality template is one of the most frequent causes of sequencing reaction problems.
DNA template quality can be affected by:
- Contaminants like salts or organic chemicals carried over from template preparation.
- Incomplete removal of cellular components like RNA, proteins, polysaccharides and contaminating chromosomal DNA.
- DNA degradation in storage.
- Multiple DNA templates in the sequencing reaction.
Contaminants
Proteins, RNA, chromosomal DNA, excess PCR primers, dNTPs, enzymes, buffer components, residual salts, organic chemicals and detergents are all considered potential contaminants for DNA sequencing. The presence of contaminants in the template preparation can inhibit the sequencing reaction resulting in weak signal strengths.
DNA degradation in storage
Repeated freezing and thawing or nuclease contamination in a template preparation can cause the DNA to degrade over time.
Multiple DNA templates in the sequencing reaction
The presence of more than one template in a sequencing reaction will cause multiple overlapping sequences in the data. This can be seen with both PCR products and cloned DNA templates. In PCR reactions in particular, more than one product can be produced when the PCR conditions are not optimised for complete specificity. Most clean up procedures for PCR reactions are designed to remove unincorporated nucleotides, salts and remaining PCR primers, not secondary PCR products. These secondary PCR products can be observed by agarose gel electrophoresis as differently sized bands from the one intending to be sequenced.
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