dGTP | dITP | dGTP | dITP |
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Figure 2. Compression artifacts can be eliminated using dITP in place of dGTP without interference by stops or other artifact bands. Shown are two severely ‘compressed’ regions of secondary structure (see arrows). The sequences run using dITP in place of dGTP are accurate and unambiguous.
A suitable nucleotide mixture containing dITP is included in the kit for use with templates prone to gel compression artifacts. To use dITP simply substitute the dITP Nucleotide Mix for the dGTP Nucleotide Mix. All other aspects of the sequencing protocol remain unchanged except that when using dITP, reduce the termination temperature from 72°C to 60°C and increase the time to approximately 5 minutes or longer (see figure 3). The use of dITP will result in less uniform band intensities, but will completely resolve even the strongest compressions. A 40% formamide gel will also eliminate almost all compressions (see ‘Denaturing gel electrophoresis’ section).
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