1min | 4min | 10min | 20min |
300 bases
200 bases
150 bases
Figure 3. Use of dITP requires longer extension times at 60°C. Shown are four sequences of plasmid pUC18 obtained using cycles with 1, 4, 10 and 20 minute extension steps in the cycles. Extension steps of
Reading farther from or closer to the primer
The termination mixes described in the protocol will typically yield sequencing data from the first base to over 500 bases from the primer. This is as much sequence as most users will be able to read using current standard electrophoresis technology. If it is desired to obtain sequence >500 bases from the primer, the dNTP:ddNTP ratios can be easily altered to shift the distribution of sequencing reaction products by adding more dNTPs to the termination reaction. Adding 3∝l instead of 2∝l dNTPs will increase the dNTP:ddNTP ratio
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