GE 33 Recording Equipment User Manual


 
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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 4-5 minutes or longer are necessary for
reading beyond 200 bases.
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 3µl instead of 2µl dNTPs will increase the dNTP:ddNTP ratio
1min 4min 10min 20min
300 bases
200 bases
150 bases